Tierärztliche Fakultät - Digitale Hochschulschriften der LMU - Teil 02/07

Thyroid parameters in tortoises and turtles frequently presented at the veterinary

practise

The present study is concerned with the examination of thyroid parameters in those tortoisespecies

frequently presented at the veterinary practise. 338 tortoises and turtles were

examined over a period of one year. The examinations included case history, clinical

examinations, diagnosis and determination of fT3, fT4, T3, and T4 levels by way of

electrochemiluminescence immunoassay and sonographic examinations of the thyroid gland

with determination of the thyroid size by measuring.

One of the first steps was to determine the thyroid hormone concentrations of fT3, fT4, T3,

T4 in 103 healthy tortoises and turtles. Subsequently the levels were examined for

dependencies regarding species, age, sex, and season. This revealed significantly higher fT4-

and T4-concentrations in turtles compared to tortoises. A diagnostical dependence regarding

age, sex, and season could not be detected. Accordingly a reference range, defined as 95%

percentile interval, was established for those tortoises frequently presented at the veterinary

practise (Testudo hermanni, Testudo graeca, Testudo marginata, Agrionemys horsfieldii).

In a second step the fT3, fT4, T3, and T4 concentrations of 235 ill tortoises were determined.

The animals were divided according to manner and severity of their illness into three groups

(slightly ill animals, profoundly ill animals, and animals under medication by Allopurinol).

Significant differences were revealed in the thyroid hormone concentrations of healthy

compared to profoundly ill animals. The main parameter for diagnostic use, it became, is

apparently fT3. There is evidence suggesting an increase of this parameter in excess of the

reference range indicates that the animals suffer from a severe illness with possibly fatal

consequences. This test has a high specificity and a comparatively low sensitivity.

At the same time the thyroid glands of 338 tortoises were examined sonographically. Position,

shape, echogenicity and echotexture were described.

The maximum diameter of the thyroid gland was established and put in relation to the body

weight. Dependencies between the size of the thyroid and the state of health or illness on the

one hand and the concentration of thyroid hormones on the other hand were checked. This

showed that animals under medication by Allopurinol frequently feature bigger thyroid glands

than healthy animals. A correlation between thyroid hormone concentration and thyroid-size

could not be proven.

The results of this study are compared to evidence from to literary references and the

diagnostical applicability is discussed.

Protein misfolding and aggregation are thought to underlie the pathogenesis of many „amyloid diseases“, such as Alzheimer and Parkinson diseases, whereby a stepwise protein misfolding process begins with the conversion of soluble protein monomers to prefibrillar oligomers and progresses to the formation of insoluble amyloid fibrils. Drusen are extracellular deposits found in eyes afflicted with age-related macular degeneration (AMD). Recent characterizations of drusen have revealed protein components that are shared with amyloid deposits. However, characteristic amyloid fibrils have thus far not been identified in drusen. In this study, we tested the hypothesis that nonfibrillar oligomers may be a common link in amyloid diseases. Oligomers consisting of distinct amyloidogenic proteins and peptides can be detected by a recently developed antibody that is thought to recognize a common structure. Notably, oligomers exhibit cellular toxicity, which suggests that they play a role in the pathogenesis of neurodegenerative diseases. Through use of the anti-oligomer antibody, we came to observe the presence of nonfibrillar, toxic oligomers in drusen. Conversely, no reactivity was observed in age-matched control eyes without drusen. These results suggest that amyloid oligomers may be involved in drusen biogenesis and that similar protein misfolding processes may occur in AMD and amyloid diseases.

This work deals with the production of antibodies against each component of the Non-hemolytic enterotoxin (Nhe) of Bacillus cereus and their use for qualitative and quantitative analyses of the enterotoxin profiles of 100 B. cereus strains.

To facilitate the detection of this toxin, consisting of the subunits NheA, NheB, and NheC, a complete set of high-affinity antibodies against each of the three components was established and characterized. A rabbit antiserum specific for the C-terminal part of NheC was produced using a respective synthetic peptide coupled to a protein carrier for immunization. Using purified B. cereus exoprotein preparations as immunogens, one monoclonal antibody against NheA and several antibodies against NheB were obtained. No cross-reactivity with other proteins produced by different strains of B. cereus was observed. Antibodies against the NheB component were able to neutralize the cytotoxic activity (up to 98 %) of Nhe.

To evaluate the expression of HBL and Nhe, a set of 100 B. cereus strains was examined. Molecular biological characterization showed that 42 % of the strains harboured the genes for HBL and 99 % for Nhe. The production of all HBL and Nhe components was analyzed using specific antibodies and, in culture supernatants, detectable levels of HBL and Nhe were found for 100 % of hbl-positive and 96 % of nhe-positive strains. The concentrations of the HBL–L2 and NheB component ranged from 0.02 to 5.6 µg/ml and from 0.03 to 14.2 µg/ml, respectively. Comparison of the amount of NheB and HBL-L2 produced by food poisoning and food/environmental strains revealed that the median values for all food poisoning strains were higher than for the food/environmental isolates.

The data presented in this work provide evidence that specific and quantitative determination of the enterotoxins is necessary to evaluate the enterotoxic potential of B. cereus. In particular, the level of Nhe seems to explain most of the cytotoxic activity of B. cereus isolates and may indicate an higher diarrheic potential than anticipated.

Derzeit existiert keine rechtlich bindende Grundlage für die Haltung von Mastputen in Deutschland. Hinreichend bekannte Probleme in der Putenmast, wie Erkrankungen des Skelettsystems und Verhaltensanomalien werden nicht selten in direkten Zusammenhang mit der Haltungsumwelt gebracht und sind im Hinblick auf vermeidbare Schmerzen und Leiden der Tiere von tierschutzrelevantem Interesse. Auch der erhöhte Anspruch des Verbrauchers verlangt nach einer Verbesserung verschiedener Aspekte in der Putenproduktion. Somit ist es notwendig, dass auch alternative Haltungssysteme untersucht werden.

Die vorliegende Studie basiert auf dem Vergleich zwischen den Putenherkünften B.U.T. Big 6 und Kelly Bronze, die auf zwei getrennten Freilandflächen unter nahezu identischen Bedingungen eingestallt und 22 Wochen lang gemästet wurden. Die Tiere wurden jeweils nicht schnabelkupiert. Im Laufe von zwei Mastperioden (Sommer 2004 und Winter 2004/05) wurden Parameter des Verhaltens, der Leistung, der Tiergesundheit, der Fleischqualität und der Knochenbruchfestigkeit vergleichend untersucht. Ziel der Untersuchung war die Feststellung, ob und in wie weit sich die Mastputenhybride B.U.T. Big 6 von der als Robustrasse geltenden Kelly Bronze Pute unter diesen Bedingungen unterscheidet.

Insgesamt gesehen lassen die in dieser Studie erhaltenen Ergebnisse den Schluss zu, dass es neben der Haltung von Robustrassen möglich ist B.U.T. Big 6 Puten tiergerecht und leistungsstark im Freiland zu mästen, obwohl gesicherte Unterschiede zwischen den Gruppen festgestellt wurden, wobei die Kelly Bronze Puten bezüglich ihrer physiologischen Voraussetzungen besser als die B.U.T. Big 6 Puten abschnitten.

Anhand immunhistochemischer, ultrastruktureller, lichtmikroskopischer und glykohistochemischer Untersuchungen wurde der Nebenhoden des Hundes untersucht.

Kim-1 (kidney injury molecule-1), a member of the TIM (T-cell immunglobulin mucin) family is a glykosylated type 1 transmembrane protein, which is markley upregulated during the repair of renal epithel cells after ischemia and in malignant and cystic kidney disease, while expressed at low levels in normal kidneys. In previous experiments Kim-1 has been shown to localize in primary cilia of cultured renal epithelial cells in-vitro. Primary cilia have been shown to play a role in development and recently cilial dysfunction has been shown to be the unifying defect causing polycystic kidney disease. Kim-1 interacts with Inversin, a ciliary protein, which is mutated in Type 2 nephronophthisis, and which has been shown to modulate the Wnt pathway by targeting Dishevelled (Dvl) for proteasomal degradation. The expression of Wnt proteins is necessary for many fundamental processes in differentiation, proliferation, polarity and adhesion. Wnts play an important role in the early embryonic development. There is only limited knowledge on the physiological role of Kim-1 during differentiation, proliferation and survival of tubular epithel cells after renal injury. The aim of this study was to characterize the role of Kim-1 in epithelial polarization and ciliogenesis of tubular epithel cells outside of pathological injury models. Additionally the role of Kim-1 in the Wnt signaling pathway was examined. Renal tubular cells were analyzed phenotypically with immunfluorescence staining. Protein expression levels were detected by SDS PAGE and Western blot. Signaltransduction activity was measured in luciferase reporter assays. Protein overexpression was achieved with transient transfection of plasmid DNA in cultured cells after calcium chloride precipitation. Gene reduction was achieved through lentivirus-mediated transduction of short interfering RNAs. Examination of immunfluorescence stained cryosections showed, that Kim-1 is localized in-vivo in primary cilia of tubular epithelial cells in mouse kidneys. Kim-1 expression levels correlate with the polarization of tubular epithel cells in-vitro. Reporter assays using TOP FLASH luciferase constructs revealed that Kim-1 reduced the Dvl-induced TOP FLASH activation and decreased steady-state levels of ß-Catenin in HEK 293t cells, suggesting that Kim-1 antagonizes the canonical Wnt pathway. Conversely Kim-1 expression increased AP-1 activity, which has been linked to the non-canonical Wnt pathway. Phenotypical characterization of two different immortalized renal epithelial cell lines after Kim-1 knock-down showed no changes in epithelial polarization, or ciliogenesis. These results suggest that Kim-1 is a ciliary protein that probably plays no active role in cell polarization or ciliogenesis, but may participate in the Wnt signaling pathway. Kim-1 may act as a molecular switch between the canonical and non-canonical Wnt pathway and thus may play a role in epithelial differentiation.

In der ökologischen Schweinehaltung führen u. a. fütterungsbedingte Darmerkrankungen zu hohen Verlustraten bei bereits über 12,0 kg Lebendmasse schweren Absetzferkeln. Tierärzte, Fütterungsexperten und Landwirte befürchten einen weiteren Anstieg dieser Verluste wenn bei Bioland ab Januar 2008, auch bei Ferkeln eine 100 % Biofütterung ohne konventionelles Kartoffeleiweiß verpflichtend wird. Deshalb werden die Entwicklung und Erprobung gesund-heits- und damit leistungsstabilisierende Fütterungsstrategien für die Öko-Ferkelaufzucht ge-fordert.

Im Öko-Versuchsstall des Landwirtschaftszentrum Haus Düsse der Landwirtschaftskammer NRW wurden deshalb an 240 Saug- und Absetzferkeln von 8,1 bis 26,6 kg LM und in einem Praxisbetrieb an 2002 Absetzferkeln von 10,2 bis 22,0 kg LM 8 Öko-Fütterungsstrategien bestehend aus 2 Saugferkelbei- (S1,S2) und 4 Aufzuchtfutter (A1,A2,A3,A4) auf Fitness- und Leistungs-Parameter geprüft. Im S1 (100 % Bio-Futter) bildeten 10 % Magermilch-pulveranteil und 10,0 % getoastete Sojabohnen und 20,0 % getoastete Ackerbohnen die Grundlage der Eiweißversorgung. An hochwertigen Energieträgern kamen 13,0 % Weizenflo-cken und 12,0 % Haferflocken zum Einsatz. Im S2 wurden neben 6,0 % Magermilchpulveran-teil, 10,0 % getoastete Sojabohnen, 10,0 % getoastete Ackerbohnen noch 5,0 % konventionel-les Kartoffeleiweiß eingesetzt. Die Anteile der hochwertigen Energieträger Weizenflocken und Haferflocken waren damit fast doppelt so hoch wie im S1.

Das A1 enthält keine getoastete Ackerbohnen, keine Weizenflocken und kein konventionelles Kartoffeleiweiß, im A2 sind 20 % getoastete Ackerbohnen, im A3 sind 22 % getoastete A-ckerbohnen sowie 22 % Weizenflocken und im A4 sind 10 % getoastete Ackerbohnen sowie 22 % Weizenflocken und 4 % konventionelles Kartoffeleiweiß enthalten. A1, A2 und A3 ent-sprechen ohne konventionelles Kartoffeleiweiß einem 100 % Biofutter.

Die Untersuchungen ergaben folgende Ergebnisse:

• Die Fruchtbarkeitsleistungen der Sauen erreichten mit 11,4 bzw. 12,1 lebend gebore-nen Ferkeln und 9,6 bzw. 9,4 abgesetzten Ferkeln jeweils pro Wurf an beiden Standorten ein gutes Ergebnis, allerdings führte die lange Säugezeit von 48 Tagen in Haus Düsse bei den Erstlingssauen zu sehr hohen Substanzverlusten von über 12 % in der Säugezeit.

• Der Gesundheitszustand der Ferkel war in Haus Düsse unbefriedigend, in allen 4 Prüfdurchgängen traten über alle Futtergruppen verteilt bereits bei Saugferkeln Durchfaller-krankungen aufgrund Coli- und Streptokokkeninfektionen sowie eines Kokzidienbefalls im 3. und 4. Durchgang auf, nach dem Absetzen erkrankten die Ferkel oftmals erneut an coli-bedingten Durchfällen in allen Futtergruppen, die anatomischen und bakteriologischen Un-tersuchungsbefunde von Sektionen lassen erkennen, dass sowohl die Haltungsbedingungen als auch das Nährstoffangebot mit den eingesetzten Prüffuttern unzureichend waren und deshalb eine weitere Verbesserung von Haltungsmanagement und Fütterungsstrategien für Ferkel und aufgrund der frühen Erkrankungen der Saugferkel auch für Sauen notwendig ist.

• Die Keimgehalte (aerobe und anaerobe Gesamtkeimzahlen, Enterobakterien, Laktoba-zillen, Cl. perfringens und Hefen) der 700 Kotproben in der 4., 8., 9. und 10. Lebenswoche in Haus Düsse und der 64 Kotproben in der 7. und 9. Lebenswoche im Praxisbetrieb lassen nur beim Gehalt an Laktobazillen tendenzielle Unterschiede bei den Saugferkelfuttern er-kennen, das S1 mit höherem Magermilchpulveranteil zu geringfügig höheren Werten.

• Die IgG-, IgM- und IgA-Gehalte am 2., 26. und 38. Lebenstag in Milch und Blut und in der 8., 9. und 10. Lebenswoche im Blut lassen bislang keine Unterschiede zwischen den Futtervarianten erkennen; die im Vergleich zu anderen Untersuchungen höheren IgG- bzw. IgA-Konzentrationen im Blutserum am 38. Lebenstag (knapp 10 mg IgG bzw. ca. 1 mg IgA je ml Blutserum) sind vermutlich auf die längere Säugezeit bei Öko-Ferkeln zurückzufüh-ren.

• Eine tendenziell höhere Leistung erreicht das mit 10 % Magermilch

The study is subdivided into two different parts: the first part deals with the development of a method to gain uterus milk in vivo during the preimplantation periode in cattle for the investigation of regulatory factors. The second part investigates different proteases in bovine follicles 20 hours after GnRH (Gonadotropin releasing hormone) injection (shortly bevor ovulation) for comparable as well as in the corpus luteum (CL) during oestrous cycle and induced luteolysis. In addition apoptotic as well as anti-apoptotic factors were evaluated in the CL during oestrous cycle and induced luteolysis.

For the development of a method for gaining uterus milk in vivo during the first 24 days of gravidity in cattle, nine heifers were cycle synchronised using the Ovsynch method and artificially inseminated. Before flushing an epiduralanaesthesia was given and both uterus horns were flushed with 13ml 0.9% NaCl using a balloon embryo transfer catheter at day 5, 7, 12, 17 and 24 of gravidity. The catheter was placed 1cm cranial to the bifurcatio uteri in both horns. It was possible to retrive between 3ml and 13ml of the used flushing fluid. The uterus milk from the ipsilateral horn was inspected for an embryo and an EDTA-stabilisator was given to the uterus milk of both horns. An infection of the uterus occured in three heifers after the second and in five heifers after the third flushing. In one heifer no infection was found. Between day 17 and day 24 all heifers showed clear signs of oestrus. It was possible to detect progesterone, oestradiol-17-beta, PGF2alpha and VEGF via enzyme immunoassay (EIA) and radio immunoassay (RIA), respectively. Because of the occurred infection no statistic analysis was made. But it could be seen that the level of progesterone ranged between <0.1 and 0.4ng/ml, whereas no correlation to the inflammation was found. The values of the native uterus milk should be about 10-fold higher, because of the diluting effect of the flushing fluid. The pattern of oestradiol-17-beta was erratic, because the oestradiol levels of the flushing fluid from heifers with no infection till day 12 of gravidity could not be related to each other. It could be shown in further tests that high oestradiol-17-beta levels were measured via EIA in 0.9% NaCl with EDTA-stabilisator compared to 0.9% NaCl without the stabilisator. VEGF revealed levels between 0.08 and 2.58ng/ml, whereas the higher levels were seen during inflammation. The level of PGF2alpha showed a clear correlation to the infection. But there were also differences seen between the different heifers or even between the two uterus horns of one animal during the first flushing, which might be due to the massage of the uterus horns while flushing, which can induce prostaglandin secretion. The method of gaining uterus milk in vivo should be changed as follows: to prevent an endometritis just one flushing should be done per gravidity with gaining of the embryo, so that the heifer can be inseminated again after two oestrous cycle. Again both horns should be flushed, whereas one sterile disposable catheter should be used for each horn and the flushing should be devided into two parts: first flushing with 13ml of sterile 0.9% NaCL for biomolecular methodes, then flushing of the embryo using 5 x 50ml sterile PBS buffer. The embryo should be frozen in RNA-Later at -80° for further investigations. The received flushing fluid should be separated in two aliquotes, whereas one is used for the oestradiol-17-beta measurement and must not contain the EDTA-stabilisator. These two aliquotes should be centrifugated and the fluid should be stored seperatly at –20° from the received cell pellet. Part of the cell pellet can be used for the identification of the cells found in the uterus lumen and the rest stored in RNA-Later at –80° for further biomolecular investigations. For the evaluation of prostaglandins in the uterus milk it should be considered to treat the heifers with a prostaglandin synth

Zum Nachweis von PrPc und PrPsc wurde eine Kapillarelektrophorese-Immunofluoreszenz-Methode (CE-LIF) etabliert und mit konventionellen Verfahren (ELISA, Westernblot, Dot-EIA) verglichen. Zur CE-LIF wurde der monoklonale Antikörper F89 und ein Fluoreszenz-markiertes Peptid (FITC-P3) eingesetzt. Die Nachweisgrenze für rekombinantes bovines PrPc liegt bei 1 pg/Test.

Zur Anreicherung von PrPsc aus Hirn und Liquor wurden (Ultra-) Zentrifugations-, Extraktions- (HFIP) und Chromatographieverfahren (HPLC, Festphasenextraktion) einzeln oder in Kom-binationen geprüft; darüber hinaus wurden Anreicherungsversuche mit Affinität- bzw. Immu-nomagnetischer Separation vorgenommen.

Die Überprüfung des PrPsc-Gehaltes ausgewählter Extrakte im Vergleich zum Ausgangsma-terial mittels ELISA ergab deutliche Substanzverluste. Eine selektive Bindung von PrPsc an Dynabeads® gekoppeltem Plasminogen konnte nicht nachgewiesen werden. Dagegen wurde eine Anlagerung an Antikörper-beschichteten Beads festgestellt. Allerdings war die Bin-dungskapazität der Beads bei Versuchen in mit in PBS gelöstem PrPc oder PrPsc höher als bei vergleichbaren Versuchen mit Liquor.

Bei der kapillarelektrophoretischen Analyse erwiesen sich alle mittels einfacher Probenauf-bereitung hergestellten Extrakte als ungeeignet. Sie führten entweder zu technischen Prob-lemen (Verstopfung der Kapillare; Durchbrennen) oder zu Interferenzen im Elektrophe-rogramm, die eine Auswertung nicht ermöglichten. Die Aufbereitung mittels HPLC ergab zwar verwendbare Präparationen, allerdings variierte die Retentionszeit von PrPsc derart stark, dass dieses Verfahren nicht weiter eingesetzt wurde. Bezüglich der Verwertbarkeit von CE-LIF wurden die besten Resultate mit Hilfe einer Aufreinigung nach Bio-Rad® mit an-schließender Festphasenextraktion erzielt. Während der Nachweis von PrPsc mittels CE-LIF aus Hirngewebe eindeutig gelang, führte die Untersuchung des Liquors eines bekannt BSE-positiven Tieres zu keinem eindeutigen Ergebnis.

Prinzipiell ist die CE-LIF zum Nachweis von PrPsc geeignet. Allerdings handelt es sich dabei um ein sehr störanfälliges Verfahren. Die Sensitivität kann deutlich erhöht werden, wenn die Konzentration von PrPsc in kleine Volumina noch besser gelingt.

Infections of the bovine mammary gland are, in addition to the affliction of the animals, a great economic burden in the dairy industry. A better understanding of the innate immune response of the host could lead to a better selection of mastitis resistant cows or to better prophylactic and therapeutical treatments. The objective of the present study was to investigate the involvement of the epithelial cells into the outcome of mastitis induced by different pathogens. Primary epithelial cell cultures isolated from milk of Brown swiss cows and coloured german goats, and tissue of cows were used to test the immune response. Because the cells of the cows showed different responses to isolated bacterial endotoxins (lipopolysaccharide, lipoteichoic acid, and peptidoglycans) compared to whole bacteria, they were treated with heat inactivated (10 MOI) gram-negative Escherichia coli (E.coli), a very common pathogen causing acute and severe intra-mammary infections, with Staphylococcus aureus (S.aureus) and coagulase-negative Staphylococcus (CNS), both prevalent cause of chronic and subclinical, and, Streptococcus uberis (Str.uberis) an inducer of acute and chronic mastitis. Goat cells were treated with 10 MOI heat inactivated S.aureus causing in opposite to cows an acute, clinical mastitis and LPS. In cows E.coli induced an increased mRNA expression of interleukin-(IL) 8, IL-6, tumor necrosis factor alpha (TNF) and granulozytes, macrophage-stimulating factor (GM-CSF) within a 30 min treatment. IL-8, IL-6 and TNF mRNA levels were still elevated after 60 min, 90 min, 6 h and 24 h. After 6 h S.aureus induced an increase in mRNA expression of IL-6, IL-8, TNF and SAA. After a 24 h treatment the expression of these immunomodulators was still elevated. Interestingly, Str.uberis in the same concentration did only induce the mRNA expression of GM-CSF after 60 min and the IL-8 after a 6 h treatment but had no influence on other immunomodulator mRNA expression. CNS treated cells needed a MOI of 50 for any reaction. This MOI induced a reaction on mRNA levels of IL-6 and IL-8 but not on TNF after 6 h. In goats S.aureus induced an increase in mRNA expression of IL-8 and TNF after 1 and 6 h. After a 24 h treatment just the mRNA level of TNF was elevated.

In conclusion mammary gland epithelial cells are involved in the different immune response to various mastitis pathogens. In cows, E.coli always induced an earlier immune response compared to S.aureus. This pathogen induced a later and probably for the immne system weaker reaction, not strong enough to cause an acute intramammary infection. Str.uberis in the same concentration induced only an increased GM-CSF and IL-8 mRNA expression after a 60 min or a 6 h treatment, respectively. This bacterium probably stimulates different immune relevant gene expressions not measured here or does not stimulate the mammary gland epithelial cells. The cells needed a 5-fold higher dose of KNS to respond with induced mRNA levels of IL-6 and TNF but not IL-8. KNS maybe does not stimulate the immune system enough to induce an acute mastitis and is like by the epithelial cells just recognized in very high dosis. IL-8 plays an important role as a chemotactic stimulus that is missing here. Goat mammary gland epithelial cells showed a early increase of IL-8 and TNF, that could be high enough, combined with the response of the immune cells in the milk, to induce an acute and severe intra-mammary infection.