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

Georg, Christine Elisabeth Structure of the ciliary body and its annexes in different bird species: a light- and scanning electron microscopic study. The aim of this study was to clarify the structure of the ciliary body and its annexes in different bird species. Therefore the ciliary body, the chamber angle, the fibrae zonulares and the iris of 52 birds of 19 different species were examined systemically for the first time, not only by light microscopy, but also by scanning electron microscopy. Vicarious for the different types of bulbi oculi were examined gallinaceous birds, day-active and night-active predatory birds. The examination showed that the general anatomy of the ciliary body is equal in all birds. It can be divided morphologically into two parts from posterior to anterior: the posterior part of the pars plicata and the anterior part of the pars plicata. A prelenticular pars plicata, pars plana and transitional region of the iris, as it exists in mammals for the production of the aqueous humour, cannot be found in birds. In the avian eye, the aqueous humour is produced by the anterior part of the pars plicata. Large species-specific differences in the content of elastic fibres in the ciliary body could be found. Gallinaceous birds, day-active predatory birds and the cormorant use lenticular accommodation. Their ciliary body therefore contains many elastic fibres, while in night-active predatory birds, which almost exclusively use corneal accommodation, the ciliary body contains almost none. The size of the different portions of the ciliary muscle depend on the kind of accommodation used in the various bird species. In gallinaceous birds and day-active predatory birds, both main parts of the ciliary muscle, the ciliaris anterior and posterior muscle are equal in size. In contrast to this, the anterior portion in night-active predatory birds and the posterior portion in the cormorant is much larger. The avian-specific active accommodation requires an extremely deep cilioscleral sinus in all bird species. The width of the chamber angle depends on the bulbus type. In gallinaceous birds it is narrow, while in night-active predatory birds it is wide open. In day-active predatory birds its width is intermediate. The pectinate ligament in the bird is not strong enough to support the structure of the ciliary body. It consists of thin, mostly parallel cords which pull the ciliary body back into a relaxed postion when the ciliaris posterior muscle relaxes. In contrast to this, in the cormorant eye, the pectinate ligament consists of a very dense network of thick fibres. The examination of the Schlemm's canal showed clearly, that birds have a sinus venosus sclerae, in contrast to the plexus venosus sclerae in most mammals. In the anterior part of the iris of the examined Phasianus colchicus, Tinnunculus tinnunculus, Hierofalco rusticolus and Haliaeetus leucocephalus epitheloid cells were found, which are similar to the pigment cells, typical to many night active predatory birds.

SUMMARY Immunological monitoring in the context of gene therapy of feline fibrosarcomas Cytokine kinetics and humoral response to vector and transgene In the context of an adenoviral gene therapy of the fibrosarcoma of the cat we introduced the human interleukin 2 (huIL 2)-gene and the feline Interferon g (feIFN g)-gene, one or the other or a combination of both. The aim of these studies is to evaluate the toxic risk and the therapeutic potential of multiple injections of two adenoviral vectors (type 5) expressing immunostimulating genes as adjuvant therapy in cats with spontaneous cancers. Several immunological parameters such as cytokine concentration, antibodies of different classes against vector and transgene and their neutralizing capacity were examined in the thesis at hand in connexion with the questions of adenoviruses in cats, pre-existing immunity and influence of inflammatory context and way of application. The kinetic of the production of IL 2 in different therapeutic approaches was evaluated. A dose-dependency was shown. The levels found in the group treated with a combination of both genes were lower than in the group treated with just AdhuIL 2. Prospects of explications are the shutdown of the viral CMV-Promoter due to the presence of interferon, the rivalry of the two transgenes to transduce a cell and the antiproliferative and antiviral effect of IFN g which directly reduces vectors and infected cells and indirectly lowers the IL 2-production by this means. More than that the application mode played an important role: The plasmic cytokine concentration was more than doubled after intratumoral injection compared to a postoperative use to the tumour’s bed. The serum peak 24 hours after vector application, which quickly redescended, is no decisive factor for the course of cytokine concentration observed locally at the tumor site. Literature shows rather a consistent local secretion for a period of nine days with intensity ten times higher than measured in blood circulation. There are indices that the serum level of IL 2 the day after the first gene transfer may be predictive for the appearance of recidivism within the first year. The tendency of infected cells in?vitro to express more IL 2 without the presence of IFN g is the same. The possibility to dose feIFN g per ELISA is presently not given. Furthermore the development of anti-huIL 2-antibodies was surveyed for a period of one year following the gene-carrying vector application. The immunoglobulins appeared early in the group of treatment with IL 2 alone (day 30), and a tardily rise of Ig G was detected in the therapeutic group with association to IFN g (day 90 to 180). By measuring the existing antibodies capable to detect or to neutralize as well the used adenovirus in ELISAs and biological assays the influence of the transgene on the antiadenoviral immune response could be proofed as well as the influence of the lieu of administration in which the vector is applied. Antiviral Ig M showed up day 14 and Ig G day 30, but their occurrence was much higher in animals treated with interferon (solitary or in association). The neutralizing capacity reached high percentages even in elevated dilutions after just one week. Moreover the immunological response to the vector depends on the application mode in terms of considerably reduced neutralizing antibodies after intratumoral injection compared to the postoperative inoculation to the tumor-surrounding tissues. The parallel-lessened defence to the transgene in this case may be assumed. Altogether the described methods are convenient to ensure an immunological monitoring of veterinary patients during an adenoviral gene therapy and to document the reactions to the applied viral vector and the expressed transgene. RESUME Surveillance immunologique du chat lors de la thérapie génique du fibrosarcome félin Cinétique des cytokines et réponse humorale contre vecteur et transgène Dans le contexte d´une thérapie génique adenovirale du fibrosarcome du chat nous avons introduit les gènes de l´interleukine 2 humaine (huIL 2) ou de l´interféron g félin (feIFN g), seuls ou en combinaison. Le but de ces études est d’évaluer le risque toxique et le potentiel thérapeutique d´injections multiples de deux vecteurs adénoviraux (type 5), contenant des gènes immunostimulateurs, comme thérapie adjuvante dans des cancers spontanés des chats. Plusieurs paramètres immunologiques comme la concentration des cytokines, des anticorps de classes différentes contre le vecteur ou le transgène et leur capacité de neutralisation ont été examinés dans la thèse présentée en rapport avec la question d´adénovirus chez le chat pour déterminer l’impact de l´immunité anti-adénovirale préexistante, l´influence d´un contexte inflammatoire et le mode d´administration. La cinétique de la production d´IL 2 dans différentes approches thérapeutiques a été évaluée. Une relation dose-réponse a été démontrée. Les niveaux trouvés dans le groupe traité avec une combinaison de deux gènes étaient plus bas que dans le groupe traité avec AdhuIL 2 seul. Ces résultats peuvent être expliqués par la coupure du promoteur viral CMV due à la présence de l´interféron dans le tissu, la compétition des deux vecteurs à transgènes différents à transduire une cellule et l´effet antiproliferatif et antiviral de l´IFN g qui limite la persistance de l’infection et engendre de ce fait une baisse de la production d´IL 2. De plus, le mode d´administration joue un rôle important: La concentration de cytokines dans le sérum a plus que doublé après une injection intratumorale, contrairement à une application postopératoire dans le lit tumoral. Le pic de concentration de cytokines dans le sérum, qui apparaît 24 heures après l´administration du vecteur et qui disparaît très vite, n´est pas un facteur décisif pour l’évolution de la concentration de cytokine observée localement au site de la tumeur. La littérature montre plutôt une sécrétion locale continue pour une période de neuf jours, avec une intensité dix fois plus élevée que celle mesurée dans la circulation sanguine. Certaines indications montrent que le niveau d´IL 2 dans le sérum, un jour après le premier transfert génétique, peut refléter les risques d’une récidive au cours de la première année. KeywordsBitte tragen Sie hLa tendance des cellules infectées in?vitro d´exprimer plus d´IL 2 sans la présence d´IFN g est la même. La possibilité de doser feIFN g par ELISA n´est actuellement pas donnée. En outre l´ apparition d´anticorps anti-huIL 2 a été surveillée pendant un an, après administration du vecteur porteur des gènes. Ces immunoglobulines se produisent tôt dans le groupe de traitement avec l’IL 2 seul (jour 30), et une hausse tardive des Ig G a été détectée dans le groupe thérapeutique avec l´association d´IFN g (jour 90 à 180). En mesurant la quantité d’anticorps capables de détecter ou de neutraliser même l´adénovirus utilisé, par des tests d´ELISA et biologiques, l´influence du transgène sur la réponse immunitaire anti-adénovirale et aussi l´influence de l´endroit de l´application dans lesquels les vecteurs sont injectés ont été démontrées. Les Ig M antiviraux se manifestaient jour 14 et les Ig G jour 30, mais étaient, en l´occurrence, beaucoup plus forte chez des animaux traités avec l´interféron (seul ou en association). La capacité de neutralisation atteint des pourcentages élevés à des dilutions importantes après une semaine de traitement. De plus, la réponse immunologique contre le vecteur dépend de la voie d´ administration: la quantité d’anticorps neutralisants est nettement réduite après injection intratumorale, contrairement à une inoculation postopératoire dans les tissus autour de la tumeur. La défense contre le transgène peut être soupçonnée d´être diminuée en parallèle. En tout, les méthodes décrites sont adéquates pour garantir une surveillance immunologique des patients vétérinaires lors d´une thérapie génique adénovirale et pour témoigner les réactions au vecteur viral appliqué et au transgène exprimé.

Angesichts der zunehmenden Nachfrage nach Geflügelfleisch von Seiten des Verbrauchers wurde die Fragestellung behandelt, ob sich Masthähnchen aus unterschiedlichen Mast-/ bzw. Schlachtbetrieben im Hinblick auf die Qualität des rohen und zubereiteten Produktes unterscheiden. Die untersuchten Broiler stammten aus vier verschiedenen Schlachtbetrieben (A bis D), die jeweils von vertraglich gebundenen Mästern beliefert wurden. Alle Schlachtbetriebe entsprachen dem EU-Standard. Bei den Haltungsformen handelte es sich um konventionelle Bodenhaltung. Die Mäster von Betrieb C verfügten über ein BTS-Haltungssystem (Besonders Tierfreundliche Stallhaltung), welches den Tieren zusätzlich den Auslauf in einem überdachten Außenbereich ermöglicht. Die geschlachteten Tiere wurden von einem nordbayerischen Unternehmen direkt vom jeweiligen Schlachtbetrieb bezogen und frisch zur Untersuchung angeliefert. Die Broiler (n = 64) wurden im Hinblick auf ihre sensorischen Eigenschaften, ausgewählte chemische Parameter, ihren Brennwert sowie ihren Gehalt an auspreßbarem Gewebewasser analysiert. Die sensorische Untersuchung wurde an ungewürzten und an mit einer vorgefertigten Gewürzmischung gewürzten Hähnchen anhand einer bewertenden Prüfung mit 5-Punkte Skale durchgeführt. Die Zubereitung erfolgte mit einem speziellen „Grillhähnchen-Programm“ in einem Combi- Dämpfer. Die chemische Analyse sowie die Bestimmung des auspreßbaren Gewebewassers wurden in Anlehnung an die Amtliche Sammlung von Untersuchungsverfahren nach § 35 LMBG durchgeführt. Zur Berechnung der Brennwerte dienten einzelne Parameter aus der chemisch- physikalischen Analyse. Nach statistischer Auswertung der Ergebnisse der chemisch-physikalischen Untersuchung, der Ermittlung des ungebundenen Gewebewassers und der Brennwertuntersuchung, anhand des Wilcoxon‘schen-Rangsummentests, zeigte sich, daß die untersuchten Broiler der Chargen C signifikant weniger Fett und signifikant mehr Rohprotein enthielten. Das auspreßbare Gewebewasser war bei Charge A signifikant niedriger als bei den übrigen Chargen. Die Brennwerte waren bei allen Chargen weitgehend deckungsgleich, allerdings enthielt C im Vergleich zu den übrigen Chargen signifikant mehr Kilojoule aus Rohprotein als aus Fett. Die sensorische Untersuchung zeigte, daß alle Chargen über einen hohen Genußwert verfügten. Die besten sensorischen Ergebnisse wurden bei der hier gewählten Zubereitungsart allerdings bei einem mittleren Fettgehalt der Broiler erzielt. Aus den gewonnenen Ergebnissen läßt sich ableiten, daß sofern sich das Rohmaterial der Grillhähnchen innerhalb eines gewissen Qualitätsniveaus bewegt, bei gleicher Zubereitung keine bedeutenden Unterschiede in der Qualität des Endproduktes zu erwarten sind. Damit kommt den bromatologischen Aspekten wie Würzung, Zubereitung und Darreichungsform eine besondere Bedeutung zu.

The aim of this study was to test the possibilities of ultrasonography in the examination of recipient animals during commercial embryo transfer in cattle. Altogether 257 animals have been synchronised by a single or a double injection of Prostaglandine F2alpha, 33 potential recipients showed spontaneous heat. On 200 of the animals actually being used as embryo recipients, a rectal palpation as well as an ultrasonographic examination of the ovaries was performed on the day of the transfer and on day 21 of the cycle. The recipients were examined again for pregnancy by ultrasound on day 35 (28 days after the transfer) and by rectal palpation on day 49. A blood sample (plasma) was taken on day 0 (oestrus), 7 (transfer) and 21, in order to mesure the progesterone concentration (P4). An enzyme-based immunological assay was used to evaluate the plasma samples for P4 concentration. The ultrasonographically defined area of the largest cross-section of the corpus luteum was significantly correlated (r = 0,22 ; p < 0,001) with the corpus luteum size evaluated by rectal palpation. There was no relation between the scoring of the corpora lutea performed by palpation and the average P4- concentration (r = 0,13 ; p > 0,05), whereas on day 7 the luteal tissue area measured by ultrasonography was significantly correlated with the P4 values (r = 0,40 ; p < 0,001). In 55 % of the recipients, a sonographically detectable cavity within the corpus luteum was found on the day of transfer. Corpora lutea with a cavity were bigger than those without ( 3,8 cm2 vs. 3 cm2; p < 0,001), however the luteal tissue area of corpora lutea with or without cavity was not significantly different (p > 0,05). On day 21, 136 animals out of 200 (69 %) having received an embryo, showed no signs of oestrus. On day 35 and 49 pregnancy was diagnosed in 50 % and 47 % respectively. The highest pregnancy rate (60 %) was obtained in the group of recipients, with the best score evaluated by rectal palpation. However, there was no significant difference in pregnancy rate between animals with different corpora lutea scores. The pregnancy rate on day 35 was 56 % in animals with a compact corpus luteum and 45 % when a cavity was present in the corpus luteum. The mean P4 value on day 7 of animals later diagnosed as pregnant or not was not significantly different (2,1 ng/ml vs. 2,2 ng/ml). 54 animals showing clear signs of heat 21 days after the last oestrus, had a P4 value of < 1 ng/ml. The area of compact corpora lutea of recipients having a P4 content of < 1ng/ml on day 21 was significantly smaller than that of animals having a P4 value > 1 ng/ml (1,5 cm2 vs. 3,5 cm2 ; p < 0,001). The average area of compact corpora lutea on day 21 (3,6 cm2 vs. 2,3 cm2; p < 0,05) was significantly bigger in pregnant than in non-pregnant animals. The percentage of presumably (day 21), respectively definitely pregnant animals (day 35 and 49), was 86 %, 65 % and 63 % respectively with fresh embryos of good quality, 57 %, 38 % and 34 % respectively with fresh embryos of poor quality and 66 %, 45 % and 44 % respectively with frozen/thawed embryos. The use of ultrasonography in the evaluation of the corpus luteum quality of recipients prior to the transfer is of limited value in commercial embryo transfer. A good oestrus detection on day 21 is useful to get the animal pregnant as early as possible; the determination of the plasma progesterone content on this day could contribute to obtain this goal. The ultrasonographic examination of the recipients for early pregnancy diagnosis (day 35 and earlier) can help, to re-use the non-pregnant animals again as soon as possible.

Detection and Differentiation of Vibrio sp. in Crustaceans, Molluscs, and Products thereof with Regard to Routine Diagnostic The purpose of this study was to reveal the contamination rate by vibrios of commercially available crustaceaens, and molluscs, and products thereof. Beside this, a simple quick routine method for the detection should be established. Between January 2001 and May 2002 the administrative „Wirtschaftskontrolldienst“ sampled in the district of Karlsruhe 100 samples of shrimps and shells either raw or heat treated, but not ready to eat, besides this heated and ready to eat food samples. These samples were tested by means of one enrichment system and two bacterial counting methods for Vibrio sp. The enrichment system was modified from the enrichment system described by ICMSF in „Microorganisms in foods – microbiological specifications of food pathogens". The modification is explained thoroughly. Strains of V. parahaemolyticus and V. cholerae were tested afterwards at the “Nationales Referenzzentrum für Enteritiserreger” in Hamburg for the existence of pathogenic potential. A total of 22 percent of the samples were positive for Vibrio; raw shells have the highest contamination rate with 50 percent, raw shrimps (35 % positive) distinctly higher than boiled shrimps (6,8 % positive). Most of the positive results succeeded using the enrichment system. The advantages of the different investigation systems are explained in detail. A potential health risk exist preferably, if improper handling of these seafood leads to an increase of the microbiological load and no or insufficient decontamination prior to eating was performed. Regarding new habits of consumption (raw sea products) and the increase of so called „other forms“ of infectious enteritis more attention should be paid to the sporadic occurrence of this microorganisms.

Autoradiographic studies on myosmine toxicokinetics in rats Myosmine, a minor tobacco alkaloid was recently detected in a wide variety of nutritional plants including cereals, nuts, cocoa and dairy products. Under acidic conditions myosmine is easily nitrosated leading to pyridyloxobutylation either directly or through the formation of N'-nitrosonornicotine (NNN). In NNN-treated rats DNA adducts releasing 4-hydroxy-(3-pyridyl)-1-butanone (HPB) are made responsible for tumours in oesophagus, oral and nasal cavities. Whereas the pharmacokinetics and metabolism of NNN have been studied in detail no such data have been available for myosmine. In the present study time-dependent distribution, binding and excretion of radiolabelled myosmine was investigated in pigmented and albino rats by quantitative whole-body autoradiography. One rat each was sacrificed by CO2 asphyxiation six and 15 minutes as well as 1, 4 and 24 hours after intravenous injection Zusammenfassung / Summary 91 of myosmine (5-3H: 1.4 µg/kg; 8.49 MBq/kg or 2’-14C: 0.27 mg/kg; 3.74 MBq/kg). Sagittal sections of 40 µm thickness obtained after deep freezing of the rats, were exposed to image plates either before or after serial extraction with solvents and water. Additionally, sections of eyes and oesophagus of untreated rats were incubated in vitro with 2’-14C myosmine. The rats killed four and 24 hours after myosmine injection were separately housed in metabolism cages for collection of urine and faeces. High concentrations of radioactivity were present six minutes after myosmine application in Harder's gland, lacrimal, salivary and accessory genital glands, nasal cavity, liver, kidney cortex, wall of the stomach fundus as well as the melanin rich parts of the eye and skin. An accumulation of radioactivity was also seen in the contents of the stomach and bladder. A similar distribution pattern was evident after 15 minutes and one hour with stronger accu-mulation in oesophagus, medulla of kidneys and accessory genital glands. An increasing labelling was observed throughout the contents of the gastrointestinal tract. Four hours after myosmine application the labelling intensity of most organs and tissues decreased conside-rably with exception of the accessory genital glands showing an extraordinary high concen-tration of radioactivity. Additionally, the oesophagus showed more marked labelling at this time. After 24 hours nearly all of the applied labelled myosmine has eliminated from the body. However, a significant labelling was still present in melanin rich tissues and in the preputial gland. The high accumulation of radioactivity in the accessory genital glands within the first four hours after myosmine injection is a new finding which has not been reported for similar compounds such as nicotine, cotinine and tobacco-specific nitrosamines. Direct determina-tion of radioactivity in the excised glands confirmed the result obtained by quantitative autoradiography. After extraction of the slices most of the radioactivity was removed from the tissues. An increased proportion of radioactivity remained in melanin rich tissues, nasal cavity, oesopha-gus, lung and liver. A distinct labelling of oesophageal mucosa as well as pigmented parts of the eyes could also be observed after in vitro incubation of slices from untreated rats. In most of the tissues, the drop of radioactivity followed a first order elimination kinetic with half lives between 0.4 hours for salivary glands and 1.2 hours for skin melanin. Within 24 hours 72 and 15 percent of the dose have been excreted in urine and faeces, respectively. The results demonstrate the suitability of saliva and hair for biomonitoring human myosmine uptake. The increased proportion of non-extractable radioactivity in the oesophagus supports our hypothesis of the possible implication of myosmine as an initiator of human oesophageal adenocarcinoma.

Im Zeitraum von Juni bis August 2001 wurde in acht verschiedenen Metzgereien im Raum München eine Gesamtzahl von 298 Proben gesammelt. 115 Tupferproben stammten von rohem Schweinefleisch und Innereien, 183 von Gerätschaften und Oberflächen, die mit Lebensmitteln, insbesondere rohem Fleisch, in Berührung kommen. Die Proben wurden auf Yersina enterocolitica untersucht. Die Nachweismethode erfolgte in Anlehnung an die ISO DIN 10273: „Microbiology – General guidance for the detection of presumptive pathogenic Yersinia enterocolitica“ und an den Vorschlag des NCFA (Nordic Committee On Food Analysis: „Yersinia enterocolitica – Detection in foods“). Unmittelbar nach der Probennahme wurde ein Direktausstrich auf Cefsulodin-Irgasan-Novobiocin (CIN)-Agar durchgeführt und die Proben anschließend für ca. drei Monate tiefgefroren. Nach diesem Zeitraum wurde ein CIN-Agar-Ausstrich nach Inkubation der tiefgefrorenen Probe in Tryptose-Soja-Bouillon (TSB) durchgeführt. Ein Ausstrich wurde nach Vorbehandlung der Über-Nacht-Anreicherung dieser Probe mit 0,25%iger KOH angefertigt. Außerdem erfolgten selektive Anreicherungen in Irgasan-Ticarcillin-Kaliumchlorat-Nährbouillon (ITC-Nährbouillon) bzw. modifizierter Rappaport-Bouillon (MRB-Bouillon) mit anschließendem Ausstrich auf CIN-Agar. Typische Kolonien auf CIN-Agar (sog. „Kuhaugen“), die sich als Urea-positiv erwiesen, wurden mit dem API 20E weiterdifferenziert. Die gefundenen Y. enterocolitica-Isolate wurden bio- und serotypisiert und die Pathogenität mit Hilfe des Kongorot-Magnesium-Oxalat (CRMOX)-Agars bestätigt. Die meisten der pathogenen Isolate (68,8%) wurden bereits nach Direktausstrich auf selektive CIN-Agar-Platten gefunden, weitere pathogene Yersinien konnten erst nach selektiver Anreicherung in ITC und MRB identifiziert werden. Pathogene Y. enterocolitica 4/O:3 wurden in sechs der acht Metzgereien aus rohen Produkten vom Schwein nachgewiesen. Insgesamt stammten 9,5% der positiven Ergebnisse aus rohem Schweinefleisch (von 95 Proben erwiesen sich neun als positiv) und 25,0% aus Schlachtnebenprodukten (Zunge, Niere und Leber; von 20 Proben erbrachten fünf ein positives Ergebnis). In einem Betrieb konnten pathogene Y. enterocolitica auch in zwei Umgebungsproben gefunden werden. Alle pathogenen Isolate gehörten zum Bioserotyp 4/O:3. Neben den pathogenen Y. enterocolitica konnten an apathogenen Yersinien drei Mal Y. kristensenii und je ein Mal Y. intermedia, Y. frederiksenii, Y. rohdei und ein apathogener Y. enterocolitica-Stamm nachgewiesen werden. Die Ergebnisse dieser Arbeit zeigen, dass pathogene Y. enterocolitica in Metzgereien vorhanden sind, wobei der Großteil der Nachweise in unbehandeltem Schweinefleisch und in Nebenprodukten der Schlachtung gelang. Der geringe Nachweis pathogener Y. enterocolitica in den Umgebungsproben ist insofern nicht erstaunlich, als die vorhergehende Reinigung und Desinfektion eine entscheidende Verminderung der Keimflora bewirkt hatte. Die meisten Y. enterocolitica-Isolate wurden ohne Anreicherung gefunden. Dies weist auf eine große Menge an pathogenen Isolaten in den untersuchten Proben hin. Damit wird deutlich, dass das Vorkommen und die Ausbreitung von pathogenen Y. enterocolitica 4/O:3 in nicht selbstschlachtenden Metzgereien ein nicht zu vernachlässigendes Problem darstellt.

In the present study, the diet of breeding sows was supplemented with 3000 mg respectively 2000 mg of vitamin C phosphate and/or with 750 mg respectively 500 mg of iron methionin daily from day 100 of gestation until birth. To investigate an effect on erythrocytes, hematocrit, hemoglobin and other parameters blood samples were taken from the sows and their piglets. In summary, the supplementation of iron methionin resulted in the rise of some parameters relevant for the orign of an iron deficiency. The supplementation of vitamin C to the diet of breeding sows resulted in increased weight of piglets at birth. In all supplementated groups, increased levels of iron in the livers of piglets could be observed when compared with the control group.

Marek´s disease virus (MDV) is a herpesvirus causing a lymphoproliferative disorder of domestic chickens worldwide. The early signs of disease like apathy, anorexy and the transient paralysis as well as the symptoms at the later stages (Neoplasia and death) causes large economical problems in the chicken industry. With the development of an effetive Vaccine, the first practical vaccine against an oncogenic disease of any type in any spieces, most of the serious problems were solved. However recent reports describing an increased virulence resulting in vaccine breaks, indicates a demand for better vaccines. To be able to enhance the prophylaxis against Marek´s disease virus, the understanding of the interaction between host and virus needs to be expanded. Interferons are cytokines with antiviral activity. On the basis of their structural and functional features they can be divided into type I (IFN-alpha and IFN-beta) and type II (IFN-gamma) interferons. Type I plays an important role during the early phase of viral infections, while IFN-gamma has a prominent role later during the infection through its immunmodulatory functions. In this study it was shown that chicken embryocellcultures responds to the infection with either an apathogenic strain of MDV (CVI 988) or a highly pathogenic strain (RB1B), with the secretion of an antiviral factor. Neutralisation with cytokine specific antibodies revealed that this activity could only be attributed to IFN-alpha but not IFN-beta. In addition, synthesis of IFN-gamma was not observed in any of the experiments. Treatment of MDV infected embryocellcultures with either recombinant chicken IFN-alpha (rChIFN-alpha) or recombinant chicken IFN-gamma (rChIFN-gamma) led to a significant reduction in plaque numbers. This antiviral effect was still observed when cultures were treated 48 hours after infection. In order to understand the functional role of the IFNs in vivo, leghorn birds were repeatedly treated with rChIFN-alpha or rChIFN-gamma for ten weeks starting on the day of hatch. The birds were then challanged on the second day of life with a highly virulent MDV-strain (vv+, EU-1). Treatment with IFN-gamma delayed the mortality to a very small degree. In contrast, IFN-alpha treatment delayed the mortality for 3 weeks. However, prevention of the disease was not achieved regardless of the used IFN-alpha dose. The functional relevance of IFN-alpha was further underscored by experiments where birds were treated with a neutralizing monoclonal antibody to ChIFN-alpha and thereafter infected with the EU-1 strain at the second day of life. Birds in this group died 3 weeks earlier than those in the untreated control group. These data shows that Interferons play a role in the progression of Mareks disease, however, regardless the IFN-dose applicated, the disease could not be prevented by the cytokines on their own.

Es wurden insgesamt 181 Besamungsdosen von 34 in der Besamung eingesetzten Hengsten untersucht. Die Motilität wurde von bis zu 7 Personen geschätzt und mit 2 computerassistierten Programmen ausgewertet. Die Membranintegrität wurde anhand der Bromphenolblau-Nigrosin und der neuen Kovács-Foote Färbung ausgewertet (letztere beurteilt auch die Integrität der Akrosom- und Mittelstückmembran). Die so gewonnenen Daten wurden mit der erzielten Trächtigkeiten der Stuten verglichen.

Determination of serum albumin and serum globulins in dependency of age in dogs The age dependency of the parameters total protein, albumin, alpha-1-globulins, alpha-2-globulins, beta-globulins, gamma-globulins and the albumin-globulin-ratio in dogs was examined in this study. Therefore 406 blood samples from clinically healthy dogs were taken. The dogs ranged in age from six weeks to 16 years and were distributed in nine groups of age. The protein fractions were determined by two cellulose acetate electrophoresis gadgets. Therefore an automatic and a semiautomatic gadget were used. Additionally, the bromcresol green method was used for the quantitative determination of albumin. Regarding the age dependency of the examined parameters the results of the used gadgets correspond to each other. The problem of imprecision of cellulose acetate electrophoresis which is described in literature was also noticed in this study; the results of the automatic gadget were more exact. For each of the abovely mentioned parameters a significant age dependency could be proofed. Total protein increased continuously and significantly during the whole life. The mean value increased correspondingly from 45.5 +/- 1.55 g/l (0 up to 6 weeks old animals) up to 66.1 +/- 4,40 g/l (more than 10 years old animals). The values of albumin determined by the bromcresol green method increased statistically demonstrable until the age of one to two years (mean values: 30.0 +/- 1.81 g/l (0 up to 6 weeks old animals), 36.8 +/- 3.15 g/l (more than 1 and up to 2 years old animals)). Afterwards no significant changes could be seen. Concerning the values of relative albumin, a lifelong, significant decrease resulted from measuring with the automatic electrophoresis gadget; the mean value decreased from 67.2 +/- 2.67 % to 56.2 +/- 3.20 %. By the following, the results and the mean values of the globulin fractions, determined by the automatic gadget, are listed: The absolute values of the alpha-1- globulin fraction decreased statistically provable from puppy age (0 up to 6 weeks old animals: 4.1 +/- 0.37 g/l) to the end of the first year of life (more than 6 and up to 12 months old animals: 3.4 +/- 0.65 g/l). The relative values of this fraction showed the same attitude from puppy age (8.9 +/- 0.57 %) up to the group of the one to two years old animals (5.1 +/- 1.11 % ). The absolute values of alpha-2-globulins increased significantly from the youngest group (0 up to 6 weeks old animals: 2.8 +/- 0.39 g/l) up to the group of the one to two years old animals (4.9 +/- 1.34 g/l) and from the group of the five to eight years old animals (4.9 +/- 1.34 g/l) up to the oldest group (more than 10 years old animals: 5.7 +/- 1.18 g/l). Between the 2nd and the 8th year of life no significant differences could be found. The relative values of alpha-2-globulins increased significantly between the youngest (6.2 +/- 0.82 %) and the oldest group (8.6 +/- 1.61 % ). For the beta- globulin fraction a lifelong significant increase of the absolute (from 6.3 +/- 0.99 g/l to 13.5 +/- 1,61 g/l) and relative values (from 13.8 +/- 1.99 % to 20.3 +/- 2.06 %) were found. However, for the age from four months up to one year of life no statistically relevant increase could be seen. The relative and the absolute values of gamma-globulins increased significantly from puppy age (1.8 +/- 0.27 g/l, 3.9 +/- 0.64 %) to the group of one to two years old animals (6.2 +/- 1.51, 9.8 +/- 2.05 %). The albumin- globulin- ratio showed a lifelong significant decrease qualifying that between the 6th month and the 5th year of life no statistically relevant changes were provable. The mean value decreased from 2.1 +/- 0.27 (0 up to 6 weeks old animals) to 1.3 +/- 0.18 (more than 10 years old animals). To investigate the influence of the different growth and ageing process and the different life expectancy of small, medium, large and giant breeds on the course of the examined parameters, groups of weight were compared in different groups of age. But distributing the dogs to groups of weight could not analyze this problem expressively.

Im Literaturüberblick der vorliegenden Arbeit wird besonders auf die ökonomische und ökologische Bedeutung der Flusskrebse, den derzeitigen Stand der Forschung die Krebspest betreffend und die aktuelle taxonomische Klassifikation des Krebspesterregers, Aphanomyces astaci, eingegangen. Ziel der vorliegenden Arbeit war es, eine zuverlässige und schnelle Methode zum spezifischen und sensitiven Nachweis von Aphanomyces astaci direkt aus Krebsgewebe zu entwickeln. Zu diesem Zweck wurde zunächst ein kommerzielles Fertigkit (DNeasy Tissue Kit, Qiagen) als geeignete DNA-Extraktionsmethode ausgewählt. Als Untersuchungsmaterial wurde die Abdominalkutikula von Edelkrebsen (Astacus astacus) eingesetzt. Anhand von Genomanalysen der ITS-Regionen (Genabschnitte, die zwischen den die ribosomale RNA kodierenden Genen liegen) verschiedener Oomycota wurden Oligonukleotid-Primer konstruiert, optimiert und auf ihre Eignung überprüft. Ausgehend von diesen Untersuchungen wurden zwei PCR-Protokolle etabliert: eine Semi-Nested und eine Nested PCR. Die Semi-Nested PCR, die sich für die Routinediagnostik von Aphanomyces astaci als wenig geeignet erwiesen hat, bietet das Potential, als Grundlage für weiterführende Studien an anderen Arten der Gattung Aphanomyces zu dienen. Die Nested PCR mit den äußeren Primern NS 166/NS 681 und den inneren Primern BO 525/BO 640 ergab mit allen 20 untersuchten Aphanomyces astaci-Stämmen ein PCR-Produkt von etwa 115 bp, das mit einer anschließenden Restriktionsenzym-Analyse mit der Endonuklease Hph I verifiziert werden konnte. Die Nested PCR zeigte sich hochspezifisch gegenüber Aphanomyces astaci, während die DNA von anderen bei Krebsen und im Wasser vorkommenden Krankheitserregern und die DNA von Wirtsgewebe nicht amplifiziert wurde, was Grundvoraussetzung für den Einsatz dieser PCR als Standardmethode ist. Die Nachweisgrenze der Nested PCR lag bei Verwendung von Plasmid-DNA bei 1,9 genomischen Einheiten und bei Einsatz von genomischer DNA aus Pilzmyzel bei 1 ag. Als Zusammenfassung 172 Aphanomyces astaci-Sporen in die DNA-Extraktion eingesetzt wurden, lag die Nachweisgrenze bei 1 Spore. Zusätzlich wurden Infektionsversuche durchgeführt, bei denen Edelkrebse mit 10, 100 und 1000 Aphanomyces astaci-Zoosporen/ml infiziert wurden. Ein positives PCR-Ergebnis konnte ab Tag 2 post expositionem (entspricht dem Tag der ersten Probenentnahme) mit der beschriebenen Methode bei allen untersuchten Tieren beobachtet werden. Schließlich wurde eine Validierung der entwickelten Nested PCR unter Einsatz von Wirtskutikula anhand 19 diagnostischer Proben unterschiedlicher Herkunft innerhalb Europas (aus Deutschland, der Schweiz, Österreich, Schweden und England) durchgeführt und die Ergebnisse mittels RFLP verifiziert. Mit der in der vorliegenden Arbeit entwickelten molekularbiologischen Nachweismethode steht ein Verfahren zur Verfügung, das eine zuverlässige Diagnose von Aphanomyces astaci bereits im Anfangsstadium der Infektion direkt aus Krebsgewebe ermöglicht und innerhalb von 12 bis 15 Stunden (Sektion, DNA-Extraktion, Nested PCR, Agarosegel-Elektrophorese und bestätigende Restriktionsenzym-Analyse) durchführbar ist. Diese Methode ist somit ein geeignetes und zuverlässiges Mittel, um in Programmen zur Seuchenbekämpfung der Krebspest (Aphanomyces astaci) eingesetzt zu werden.

The aim of this work was to describe the importance of the estimation of the quality of cattle embryos in embryo transfer as well as to discuss the causes of the appearance of the variable embryo qualities after superovulation and the different possibilities to assess the embryo quality. After superovulation there are always variable embryo quality observed. The reasons for this are versatile. An important, the result of a superovulation treatment influencing factor, is the donor cow. Both the individual disposition, the age and as well healthy status and the stressexposition of the animals are playing an important role. The ovary reactions are rather essentially influenced by the existence of a dominant follicle at the moment of introduction of a superovulation treatment. By removal of dominant follicle the results can significant be improved. Another important factor is the sort of a gonadotrophin used for superovulation. The reaction to a superovulation treatment is not just depending on the selection of the gonadotroph hormone, but also on the composition of the preparations, the doses und the way of application. Because the morphological evaluation of embryo quality is subjected to subjective influences, it should attach importance to the training of the evaluating persons, to receive comparable results. The importance of assessment of embryo quality to select embryos is considerable. The results of embryo transfer, the pregnancy rates, are essentially depending on the quality of the transferred embryos. The survival of embryos after kryopreservation is also influenced by the embryo quality. Embryo quality can be evaluated by different approaches: if the selected embryo has to be transferred, the technique to estimate embryo quality must not be invasive. In the practice of the embryo transfer embryo quality is estimated before transfer by gross embryo morphology. However, this method is a rather subjective possibility for assessment of the embryo quality. More accurate date can be obtained by the use of invasive techniques. Metabolic tests to evaluate embryo viability include measurement of nutrient uptake, energy metabolism and oxygen uptake. Enzyme leakage and hormone or growth factor production can also give more details on the quality of embryos. Techniques which might partially are able to affect the embryo are the determination of the freezing resistance and vital staining. With vital staining, membrane integrity, which is critical for embryo survival, can be assessed by means of fluorescent probes. The most common staining methods for evaluating embryo quality are DAPI and FDA. These two fluorescent probes do not alter the embryo, so that you can still transfer the embryo after these examinations. Invasive assessment of the embryo quality mostly involves a kind of fixation or staining of the embryo and so they are not suitable for the practical use of the embryo transfer. Cytogenetic analysis of embryos is an important issue for caryotyping. Chromosomal deviations are known to cause early embryonic mortality in cattle. Determination of the total cell number and the allocation of these cells to the inner cell mass (ICM) and trophectoderm (TE) after differential staining of these cells give more details on the quality of an embryo.