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

Aetiology, signs and prognosis of male cattle with urethral occlusion The objective of this prospective study was a detailed description of the clinical signs of cattle with urethral occlusion and to define prognostic indicators. Male cattle admitted to the clinic were included in the study if their history or initial clinical examination suggested urethral occlusion. A total of 126 cattle one month to two years old were enrolled. For the description of the clinical picture the relevant anamnestic information as well as the clinical and laboratory findings are described. Prognosis was evaluated on the basis of clinical, laboratory and post mortem findings, as well as on the subsequent course of survivors. The exact cause of the occlusion was determined by post mortem examination in non-survivors. Urethral occlusion can be diagnosed by physical examination. Failure to pass urine in animals with a distended urinary bladder or subcutaneous oedema or intraperitoneal urine accumulation are considered to be diagnostic. Not only the parameters of the special but also of the general physical examination turned out to be important in the evaluation of the stage of the disease and the detection of inflammatory processes. Initially a sawhorse stance, agitation and colic were prominent, whereas animals in the advanced stage became weak and recumbent; some of them did so after a transient period of apparent improvement of their general condition. For cattle with concomitant inflammatory processes an arched back with a distinctly depressed behaviour was significant. In addition these animals were mostly in a poor nutrional state and had a dull or shaggy haircoat. Among the findings of the special physical examination, inspection and/or palpation of the penis (evidence of a penis haematoma), the ventral abdomen (evidence of oedema), the abdomen (evidence of an intraperitoneal urine accumulation and/or peritonitis) and finally the evaluation of the umbilicus (evidence of ascending infections), bladder, and kidneys were found to be meaningful. Abdominocentesis was performed, if intraperitoneal urine accumulation and/or peritonitis was suspected. Depending on the age a rectal examination was often impossible. Deep palpation of the abdominal cavity couldn’t be performed in animals with a fully developed rumen, increased tension of the abdominal wall and those with ventral oedema or intraperitoneal urine accumulation. In these cases the diagnostic importance of ultrasonography in the examination of intraabdominal structures as well as the determination of increased inflammatory alteration of the intraabdominal fluid was evident. Among the laboratory findings, only azotaemia was significant, whereas other parameters didn’t show any consistent tendency with regard to the stage of disease or the involvement of inflammatory processes. A total 42 out of 60 animals with uncomplicated urethral occlusion were discharged. Out of 18 euthanized animals 11 were put to sleep because of extensive inflammatory alterations, five because micturition was not restored post operatively, and two because of other critical diseases. 41 of the patients showed intraperitoneal urine accumulation in varying extents with infections and accompanying irreparable inflammatory alterations present in 23 cases. Out of 18 cattle with non-infected intraperitoneal urine only seven could be cured, the others were put to sleep for other reasons. In two animals with slight oedema resorption took place within three days, whereas the massive infiltration of urine into the subcutis resulted in necrosis of the affected areas and was followed by gangrene in 13 cases. Except for two animals with an uracheal abscess and a bilateral pyelonephritis respectively all were discharged after an extended stay at the clinic. By contrast, four cattle with infected oedema developed cellulitis with distinct deterioration of their general condition and had to be destroyed. Six patients were put to sleep after the initial examination or during surgery. Therefore the further progression of the uric oedema couldn’t be evaluated. In five of these cases euthanasia was a consequence of urethral occlusion, in one case it was in consequence of another disease. Thus prognosis is mainly influenced by duration of illness as well as the involvement of inflammatory processes. Animals at the early stage of the disease, but also those with uric oedema in case of uncontaminated urine have a favourable prognosis. Prognosis is doubtful in animals with peritoneal accumulation of uninfected urine. It is nearly hopeless in the case of infiltration of infected urine into the subcutis (cellulitis) or in the abdominal cavity (peritonitis). Therefore, the presence of inflammatory processes should be ruled out as much as possible before surgical intervention is considered.

Viral agents isolated from 18 snakes (families Colubridae, Viperidae, and Crotalidae) showing respiratory symptoms and neuronal diseases were investigated. Identifying the isolates as paramyxoviruses showing characteristic cytopathic effect, was carried out by electronmicroscopy and RNA-PCR of two partial genes (L and F). After sequencing RNA-PCR amplicons, identical sequences were found for the reptilian paramyxoviruses (RPMV) isolated in the same outbreak from different snake species, suggesting they are not host-specific. Sequence alignment of partial L and F genes within the RPMV group revealed sequence homology of at least 79 % for nucleotides (nt), and 94 % for amino acids (aa). Further sequence alignment included the Fer-de-Lance virus (FDLV) as a RPMV representative, the established type species of the paramyxovirus family. The greatest similarity was found to the partial L gene of Sendai virus, with 56 % nt and 61% aa identity. Consequently, the RPMV form a closely related, if somewhat diverse, group, belonging to the family Paramyxoviridae. This result was confirmed by phylogenetic analysis. The RPMV group formed three clusters for the L gene sequence and corresponding clusters for the F gene sequence, leaving the Sendai virus as an outgroup, which was included as a representative of the paramyxovirus family. Another phylogenetic analysis based on partial L and F protein sequences included the FDLV and the established paramyxovirus type species. In this case, the FDLV showed the closest relationship to the Sendai virus. The fusion (F) gene and protein of the reptilian paramyxovirus isolated from the snake Gonosoma oxycephala were also investigated. The sequences of the 1638 nt F gene and its 546 aa F protein were obtained and these sequences were compared to the F protein of the established paramyxovirus type species. The F protein, like its counterparts, was predicted to be glycosylated and to contain a furin cleavage site in the F1 und F2 subunit. Domains were predicted to be the signal-peptide (SP), a hydrophobic fusion peptide, two heptad repeats (HR A and HR B), a leucine zipper motif, and a transmembrane anchor (TM). To further characterize the F protein and identify antigenic determinants, an E. coli expression system and four plasmids containing four various constructs of the F gene were used: the first contained an insert with the entire gene; the second, an insert with the F gene minus the SP and TM; a third construct was inserted with the F2 subunit minus the SP; and the fourth contained an insert with the F1 subunit minus HR B and TM. To monitor expression, an anti-His tag monoclonal antibody in a Western blot (WB) was used. Antigenic epitopes were identified in WB by the use of polyclonal rabbit antiserum, raised against our isolate. No expression was detected for the first construct containing the entire F gene, but all other constructs produced proteins of the predicted molecular weights. The anti-viral rabbit serum detected only the protein encoded by the second construct, that included the predicted furin cleavage site. After treatment of this recombinant protein with furin, the antiserum reacted with only one of the fragments. The reactive fragment was identical to the fourth recombinant protein except for the inclusion of an additional 30 amino acids that contained HR B. The results indicate that HR B of the reptilian paramyxovirus F protein contains a linear antigenic epitope.

We describe the isolation and characterization of an iridescent virus from commercially produced colonies of Gryllus bimaculatus in Germany, which showed apparent mortality. Transmission electron microscopy studies on adult cricket specimens revealed the paracrystalline assembly of icosahedral virus particles in the cytoplasm of hypertrophied abdominal fat body cells. The infecting agent could be cultivated in the lepidopteran cell line sf-9, where it caused cytopathogenic effects such as cell hypertrophy, cytoplasmic vacuolization, and cell death within 8 days postinfection. Negatively stained virus particles (n = 100) had dimensions of 172 +/- 6 nm (apex to apex) and 148 +/- 5nm (side to side). SDS-polyacrylamide gel electrophoresis of virus proteins showed more than 20 distinct polypeptides with a major species of approximately 50 kDa. Analysis of the restriction fragment length profiles from digestion of purified viral DNA with the endonucleases EcoRI, BamHI, and HindIII showed marked differences from the profiles of iridoviruses of lower vertebrates (genus Ranavirus), e.g., Rana esculenta Iridovirus and Frog virus 3. Restriction enzyme digests with the endonucleases MspI and HpaII indicated the lack of methylation of viral DNA. Polymerase chain reaction led to the amplification of a 420-bp gene fragment with 97 % sequence homology to the major capsid protein gene of Invertebrate iridescent virus 6 (IIV-6). The data indicate, that this new isolate, which is the first iridescent virus reported from Germany, belongs to the genus Iridovirus of the family Iridoviridae. It is proposed to be termed Gryllus bimaculatus iridescent virus (GbIV). The investigation of four other iridescent-like viruses which were obtained in 1998, 1999 and 2000 from various tissues of three reptile species, e.g. Pogona vitticeps, Chamaeleo quadricornis, and Chamydosaurus kingii, revealed cytoplasmic desoxyriboviruses with the exact morphological and molecular biological characteristics of the formerly described GbIV. We conclude that all five isolates of iridescent-like viruses from Germany are identical and represent variants or strains of the type species of the genus Iridovirus, IIV-6. The data indicate, that GbIV, an invertebrate iridescent virus, is able to replicate in reptiles.

Caliciviren sind kleine, unbehüllte Viren mit einem RNA-Genom positiver Polarität. Die Vertreter der Familie Caliciviridae sind Erreger wichtiger, zum Teil sehr unterschiedlicher Erkrankungen bei Mensch und Tier. Das Feline Calicivirus (FCV) gilt als einer der Haupterreger des Katzenschnupfenkomplexes. Das FCV ist für Replikationsstudien an Caliciviren besonders gut geeignet, da es sich effizient in Zellkultursystemen vermehrt. Da der Replikationszyklus nur zum Teil aufgeklärt ist, wurden in der vorliegenden Arbeit zwei Nichtstrukturproteine des FCV, das Vpg und die RNA-abhängige RNA-Polymerase (3D-Polymerase), näher charakterisiert. Die 3D-Polymerase ist für die Replikation der viralen RNA zuständig. Das Vpg ist ein viruskodiertes Protein, welches kovalent an den 5'-Terminus der genomischen und subgenomischen RNA gebunden ist. Gegen beide Proteine wurden zunächst erfolgreich polyklonale Antikörper generiert. Untersuchungen zur Expressionskinetik ergaben, dass die 3D-Polymerase in einer frühen Phase des Replikationszyklus exprimiert wird. Sie tritt auch beim FCV-Isolat KS20 nur in Verbindung mit der 3C-Protease als sogenanntes 3CD-Vorläuferprotein auf, freie Polymerase ist nicht nachweisbar. Im Gegensatz zur 3D-Polymerase liegt das Vpg-Protein in einer späteren Phase des Replikationszyklus des FCV in freier Form vor. Es wurden für das FCV-Isolat KS20 zwei mögliche Vorläuferproteine von etwa 43 bis 45 kDa und von 25 kDa detektiert, deren Funktion unklar ist. Die 3D-Polymerase und das Vpg wurden nur im Zytopasma der Wirtszelle lokalisiert. Beide induzieren keine neutralisierenden Antikörper. Ferner beschäftigte sich diese Arbei mit der Hypothese, dass das Vpg bei der Transkription der viralen RNA eine Rolle spielt und möglicherweise als Primer für die 3D-Polymerase dient. Zum anderen wurde untersucht, ob das Vpg an der Enkapsidierung der genomischen und subgenomischen RNA in die viralen Partikel beteiligt ist. Zum Nachweis potentieller Wechselwirkungen zwischen den einzelnen Proteinen wurde eine Co-Immunopräzipitation durchgeführt. Bei keinem der untersuchten Proteinpaare konnte eine Interaktion festgestellt werden.

Insulin-like growth factors (IGF-I and IGF-II) are expressed in many cell types and tissues and act in endocrine, autocrine or paracrine manner to regulate cellular proliferation, survival and differentiation. IGF actions are initiated upon binding to the type I IGF receptor (IGF-IR) and are modulated through interactions with a family of six secreted IGF-binding proteins (IGFBP-1 to -6). Although the six conserved IGFBPs are structurally related, each of them has specific characteristics and may have specific functions. Most knowledge about the IGFBPs has been gained from the numerous in vitro studies, their specific roles in vivo are largely unknown. Transgenic mice overexpressing a particular IGFBP allow us to investigate the specific functions of the corresponding IGFBP in vivo. To this end, IGFBP-4- and IGFBP-6-overexpressing models were established and analyzed in the present study. First, an expression vector containing the murine H-2Kb promoter and a human beta-globin splicing cassette was used to construct the transgenes, to obtain ubiquitous expression of the mouse Igfbp4 and Igfbp6 cDNA. Two lines of H-2Kb-mcIGFBP-4 and ten lines of H-2Kb-mcIGFBP-6 transgenic mice were generated. The transgene was ubiquitously expressed at RNA level in both transgenic models, however, at protein level, transgene expression was only detected in the spleen, thymus, lung and kidney of both H-2Kb-mcIGFBP-4 transgenic lines, but in no organ of H-2Kb-mcIGFBP-6 transgenic mice. Phenotypic analyses of the H-2Kb-mcIGFBP-4 transgenic model revealed that overexpression of IGFBP-4 had no significant effect on the postnatal body and organ growth, except that the weight and volume of thymus in 8- and 12-week-old transgenic mice were significantly reduced (p < 0.05) compared to the controls. Histomorphometric analysis demonstrated that the volume of the thymic cortex was significantly decreased in transgenic mice (p < 0.05), whereas that of the thymic medulla was not changed. The fractions of various cell types in the bone marrow, thymus, spleen, lymph node and peripheral blood were determined by flow cytometry. No significant difference was found between transgenic and control groups, suggesting that IGFBP-4 excess in the lymphoid organs did not affect the development of the lymphatic cells. The proliferative capacity of the splenocytes of transgenic animals was significantly reduced after Con A and LPS stimulation (p < 0.05), but not altered after the stimulation by anti-CD3 and anti-IgM/IL2. This is probably due to transgenic IGFBP-4 expression restricted in the non-lymphatic cells. However, detailed expression of the transgene warrants further investigation. In order to realize IGFBP-6-overexpressing mice, a second construct was designed, namely CMV-mgIGFBP-6, in which the mouse Igfbp6 genomic sequence was cloned under the control of the cytomegalovirus (CMV) promoter. Four independent lines of transgenic mice were generated. Transgene expression was high in the exocrine pancreas and relatively low in the lung and liver. The activities of serum IGFBPs were not different between transgenic mice and controls. In transgenic mice, high levels of active IGFBP-6 were detected in the luminal content of the duodenum, but neither in the luminal contents of other segments of the gastrointestinal tract (GIT), nor in tissue extracts of all GIT segments. Glucose homeostasis was not altered by IGFBP-6 expression. Postnatal body and organ growth was not affected in transgenic mice, except for the absolute and relative weight and length of duodenum which were significantly reduced in 4-month-old transgenic mice as compared to controls (p < 0.05). This reduction was mainly due to a significantly smaller volume and surface area of the tunica mucosa as determined by histomorphometric analsis. Our analysis of the first IGFBP-6 transgenic mouse model provides direct evidence for inhibition of intestinal growth by luminal IGFBP-6 excess. This finding is important in the context of neonatal intestinal growth of mammals, considering the fact that milk contains large amount of IGFBPs which may at least in part arrive intact in the intestine.

Studies on the genetic variability of recent isolates of porcine parvovirus The aim of this study was to review the antigenetic variability of porcine parvovirus and to examine the role of porcine parvovirus in reproductive failure. Organ specimens of 500 fetuses and stillborn piglets from a total of 172 farms were analysed. Total DNA was isolated from the explants and quantitative as well as qualitative PCR was subsequently carried out. In 82 samples (15,8%) out of 28 farms (16,3%), PPV was diagnosed. In this study, we were able to show that rectal swabs are suitable substrates from which to detect porcine parvovirus in sows using a PCR analysis. In 12 out of 19 rectal swabs PPV genome was amplified. In one PPV-positive sample the sow was infected a minimum of 45 days before the rectal swab was collected. An attempt was made to cultivate porcine parvovirus out of 22 PPV-positive organ specimens of different origin. However, only 5 isolates could be cultivated and the production of progeny virus was very low in two of these isolates. Although multiple passages were performed, the virus yield did not increase. As the viral capsid is the target of host protective immunity against PPV, the complete gene of the structural protein of 7 field isolates was amplified and sequenced. The obtained sequences were aligned with the porcine parvovirus-strains NADL-2, Kresse, one challenge-virus of the year 1986 and with the vaccine strain of IDT. The sequence homology among the isolates sequenced is very high (on the level of nucleotide acids minimum 98,7%, on the level of amino acids minimum 97,7). Phylogenetic analysis revealed that there is only a distant relationship between NADL-2, Kresse and the challenge-virus on the hand and the field isolates on the other hand. A comparison of the aligned PPV-sequences from field isolates with the published sequence of two primers recommended for diagnostic PCR revealed single nucleotide differences. Therefore, it appears possible that the primer sequences published may not be suitable for a diagnostic PPV-PCR in the field. Taken as a whole, these data suggest that the porcine parvovirus may have altered antigenetically. Furthermore, the obtained sequences from field isolates of PPV now permit the examination of the antigenetic consequence of this evolution to develop effective vaccines against the PPV strains now present in the field.

About the occurrence of Transmissible Spongiform Encephalopathies and bacterial CNS infections in roe deer, red deer and chamois in Bavaria Brain samples of 849 wild ruminants (654 roe deer, 189 red deer and 6 chamois) from Bavaria were examined for the occurrence of TSE and encephalopathies caused by bacteria, respectively. For this, the following investigations were carried out: • General bacteriological investigation (aerobic incubation on blood, standard I nutrient and Gassner Agar) • Detection of Listeria spp. by selective procedures - Cultural isolation and biochemical identification with the BBL crystal ID system - Detection of the iap gene using real time PCR (Lightcycler) • Detection of PrPres by ELISA technique (BioRad) • Histological investigation - HE staining - Immunohistochemistry (mAb L42) Using conventional bacteriological methods, 464 different bacteria were isolated. 229 of them could be differentiated at the genus level and 235 at the species level. Totally, 35 different bacteria species were isolated, most frequently Micrococcus spp., Bacillus spp. and E. coli. Listeria spp. were detected in 55 brain samples (49 from roe deer, 5 from red deer and 1 from chamois). 52 isolates were identified with the BBL crystal ID system as Listeria monocytogenes and 3 as Listeria grayi ssp. murrayi. As the iap gene was detected in all strains, the correctness of the BBL result concerning L. grayi ssp. murrayi must be scrutinised. Analysis of the geographical distribution of the Listeria findings indicate a regional aggregation in Unterfranken (prevalence for roe deer: 17,8 %, versus 6,6 % in Oberbayern-Schwaben, 6,1 in Niederbayern-Oberpfalz and 0 % in Oberfranken-Mittelfranken). The histological investigation (HE staining) of 87 tissue samples contaminated with encephalitis relevant bacteria showed inflammation of different severity (e. g. perivascular infiltration (n = 26) or (meningo)encephalitis (n = 13)) in 41 cases. The validation of the ELISA technique for detecting prionprotein from wild ruminants pointed out that the examination of brain of wild ruminants lead to lower extinctions than that of bovine brain samples. Due to the fact that brain homogenate of 2 known CWD positive wapitis showed definite positive results, all samples were analysed with this test: There were no indications for the occurrence of TSE in any sample. In addition, immunohistochemical investigations of 10 % of all samples did not show PrPres. Due to the large number of samples, the prevalence of TSE can be quoted under 0,5 % for roe deer and under 1,5 % for red deer with a certainty of 95 %.

The aim of this work was the evaluation of literature about physiology of reproduction, assisted reproduction technologies and associated biotechniques in domestic dogs (Canis familiaris). In the bitch preovulatory follicular luteinization results in exposure of oocytes to increasing concentrations of progesterone 1-2 days prior to ovulation. Ovulation occurs approximately two days after the LH-peak. The bitch ovulates primary oocytes with intact germinal vesicle, which requires 2-5 days for the completion of the meiotic divisions within the oviduct. Artificial insemination (AI) in bitch can be performed either intravaginally or intrauterinely with fresh, chilled or frozen-thawed spermatozoa. Intrauterine insemination (IUI) may be carried out surgically by laparotomy or laparoscopy or non-surgically using transcervical cathetherization. In the bitch, IUI results in a high whelping rate and litter size comparable to those after natural mating. Cryopreservation of dog semen has been successfully accomplished and a variety of extenders, freezing and thawing protocols have been published. AI with cryopreserved semen generally yields lower pregnancy rates if intravaginal deposition of semen is used. A reliable method for synchronization and induction of a fertile oestrus cycle as well as superovulation by hormone treatment are not available. Canine oocytes may resume meiosis spontaneously in vitro, although at a much lower efficiency than in most other domestic species. In vitro maturation (IVM) of oocytes results in 20 to 70 % oocytes entering germinal vesicle breakdown (GVBD). Only 10 to 40 % oocytes progress to metaphase I to II. It has been shown, that cumulus morphology, stage of estrous cycle, oocyte size, cumulus-oocyte communication through gap junctions, age of oocyte donors, and serum supplementation of the culture medium influence the efficiency of IVM. Dog oocytes cultured within advanced preantral and early antral follicles in vitro are competent to resume meiosis and mature to the metaphase stage. The developmental potential of these oocytes was comparable to isolated cumulus oocyte complexes. The optimal culture conditions required for induction of capacitation and acrosomal exocytosis of canine sperm are yet to be established. Dog spermatozoa are able to penetrate the zona pellucida and the vitellus of homologous oocytes irrespective of the oocyte maturation stage. The developmental potential of fertilized dog oocytes in vitro is very low. Only one case of development to the blastocyst stage after in vitro fertilization (IVF) has been reported. The surgical transfer of ex vivo collected dog embryos resulted in birth of live puppys although the success rates were low. Up to date no reports of production of live pups after IVF from in vivo or in vitro matured dog oocytes exists. In one study three conceptuses were identified by ultrasonography twenty days after transfer of in vitro fertilised oocytes but no further development could be observed. Reliable protocols for cryopreservation of dog embryos have yet to be developed. Until recently, there has been limited interest in assisted reproduction techniques in the dogs. The rising significance of dogs as companion animals as well as interest in comparative aspects with wild-life canides will stimulate research in the fields of in vitro production of embryos, cryopreservation and embryo transfer of embryos.

The aim of this study (by means of a field test) has been to compare RODAC plates with two rapid test methods under routine conditions.

In the mare, the practical use of assisted reproduction techniques has been very limited so far. Embryo transfer is used only in a few countries to a low extent. The main reason for this situation is that there is no practical method available for superovulation in the mare. The majority of equine embryos are therefore collected from spontaneously ovulating mares. Synchronisation of the oestrous cycle of the donor and recipient mare before transfer is of major importance. In synchronized recipients, ovulation should occur one day before to three days after the donor mare's ovulation. Ovariectomized, hormone-treated mares have also been used as embryo recipients. In young, fertile mares embryo recovery rates up to 90 % have been reported. Embryo recovery rates for older, infertile mares are only between 20 to 40 %. Embryo recovery is generally performed nonsurgically by transvaginal flushing of the uterus. Nonsurgical recovery of embryos is not possible before day 6 post ovulation. Equine embryos are transferred by surgical flank incision or by nonsurgical, transvaginal transfer. Surgical transfer is mainly used in commercial embryo transfer programs with pregnancy rates up to 80%. The advantage of nonsurgical transfer is the noninvasive technique and the low costs, but results vary tremendously. Cooled, transported embryos offer great advantages in the management of transfer programs. Cooling for 24 to 30 hours does not seem to impair the viability of embryos. Conventional methods of cryopreservation or vitrification can be used for long term storage of equine embryos. Smaller embryos withstand cryopreservation better than large embryos. Micromanipulation of equine embryos can be used to produce monozygotic twins and for embryo sexing. The efficiency of embryo production by extracorporal fertilization in the horse is very low so far. Equine oocytes can be recovered by one of four methods: surgically, by transcutaneous flank puncture, by transvaginal aspiration or by aspirating, scraping or rupturing follicles of slaughterhouse ovaries. Different culture media can be used for in vitro oocyte maturation. After 30 hours of incubation in TCM 199, the maturation rate of equine oocytes to metaphase II is up to 80 % . In vivo fertilization of oocytes can be achieved by surgical transfer into the oviduct or follicles of inseminated recipient mares. The procedure of gamete intrafallopian tube transfer (GIFT) involves transfer of oocytes and spermatozoa into the oviduct of a recipient mare. Since only low sperm numbers are required, GIFT can be used for subfertile stallions, frozen-thawed semen and sexed spermatozoa. Conventional methods or vitrification can be applied for the cryopreservation of equine oocytes. Maturation rates in vitro are lower for frozen-thawed than for fresh oocytes but results of in vitro fertilization rates are comparable. In contrast, embryonic development rates of frozen-thawed oocytes seem to be affected by cryopreservation. In vitro fertilization of matured oocytes can be performed by co-incubation of oocytes and spermatozoa or by assisted fertilization techniques such as mechanical manipulation of the zona pellucida, zona drilling or intracytoplasmic sperm injection (ICSI). Fertilization rates with conventional methods are low (0-25 %), whereas fertilization rates up to 52% are reported with ICSI. Viable offspring has been produced with both techniques. Fertilized oocytes can be cultured to the stage of morula or blastocyst in vivo or in vitro. Co-culture with somatic cells seems to be important for embryonic development in vitro. Live foals have already been produced using in vitro cultured embryos, which developed from ICSI of in vitro matured oocytes.

Vasopressor Agents or Fluid Resuscitation for the Treatment of Uncontrolled Hemorrhagic Shock, an Experimental Approach in a Porcine Liver Trauma Model The standard approach to the traumatized victim in uncontrolled hemorrhagic shock is to infuse large amounts of crystalloid and colloid fluids as early, and as rapidly as possible. The aim of this strategy is to restore intravascular volume immediately and to maintain vital organ perfusion. The results of many laboratory studies and one clinical trial question the effectiveness of this treatment recommendation and suggest that in the case of uncontrolled hemorrhage aggressive fluid resuscitation may even be harmful. Hence, we evaluated the effects of fluid resuscitation vs. epinephrine vs. vasopressin vs. saline placebo on hemodynamic variables, regional organ blood flow, and short term survival in an animal model of uncontrolled hemorrhagic shock. Twenty-eight anesthetized pigs were subjected to a severe liver injury, which resulted in an average blood loss of 35 mL/kg within 30 minutes. When mean arterial pressure was < 20 mmHg, and heart rate declined progressively, the pigs were randomly assigned to receive either 1000 mL lactated Ringer´s solution and 1000 mL of gelatine solution (n=7), or an intravenous bolus dose and continuous infusion of 45 µg/kg epinephrine (n=7), or 0.4 IU/kg Vasopressin (n=7), or saline placebo (n=7). At 30 minutes after therapeutic intervention all surviving animals have been fluid resuscitated and further blood loss has been controlled surgically. Mean ± SEM arterial blood pressure at 5 minutes after therapeutic intervention was significantly (p< 0.0001 for vasopressin vs. epinephrine vs. placebo and p< 0.04 for vasopressin vs. fluid resuscitation) higher after vasopressin vs. fluid resuscitation vs. epinephrine vs. saline placebo (58 ± 9 vs. 32 ± 6 vs. 19 ± 5 vs. 7 ± 3 mmHg; respectively). Although vasopressin improved regional organ blood flow, this effect did not result in further blood loss stemming from the liver injury (vasopressin vs. fluid resuscitation vs. epinephrine vs. saline placebo 10 minutes after intervention, 1343 ± 60 vs. 2536 ± 93 vs. 1383 ± 117 vs. 1326 ± 46 mL; respectively; p

Bei diffusen Magenkarzinomen treten in 50% aller Fälle Mutationen im Gen für das Adhäsionsmolekül E-Cadherin auf. Bei einem Fünftel dieser Mutationen ist Exon 9 deletiert, was die Expression einer mutationsspezifischen Proteinstruktur zur Folge hat (d9-E-Cad). Gegen diese tumorspezifische Bindungsstelle wurde ein monoklonaler Antikörper entwickelt(d9MAk). Dieser Antikörper wurde mit Wismut-213 (Bi-213) gekoppelt, einem hochenergetischen alpha-Emitter mit einer Reichweite von 80 µm im Gewebe, einem hohen linearen Energietransfer von 100 keV/µm und einer Halbwertszeit von 45,6 min. Um das therapeutische Potential des lokoregional applizierten Bi-213-Immuinkonjugats bei disseminierter Tumorzellaussaat gegen die Nebenwirkungen auf den Organismus beurteilen zu können, wurde ein Modell der Peritonealkarzinose bei Nacktmäusen durch intraperitoneale Inokulation von humanen Magenkarzinomzellen mit d9-Cad-Expression entwickelt. Sonographie und Kernspintomographie zeigten für die Visualisierung abdominaler Tumormanifestation und posttherapeutischer Tumorregression eine unzureichende Auflösung, während die In-vivo-Fluoreszenzanalyse "Optical Imaging" für diese Anwendungen erste, viel versprechende Ergebnisse lieferte. Diese Darstellungsmethode könnte in weiteren Studien für die Peritonealkarzinose optimiert werden. Mittels Biodistributionsstudien wurde die spezifische Bindung des Bi-213-d9MAk an den Tumor sowie die Verteilung im Organismus im Vegleich zu einem unspezifischen Kontrollantikörper untersucht. Die Bioverteilung zeigte neben einer hochspezifischen Bindung des Bi-213-d9MAk in Tumoren mit d9-E-Cad-Expression niedrige Konzentrationen des Radioimmunkonjugates im Blut und in den Organen. Bei vorliegendem Aszites blieb ein Großteil des Bi-213-d9MAk abdominal retiniert. Die Überlebenszeiten der Tiere nach Inokulation von 1x10E7 Tumorzellen wurden in Abhängigkeit von der applizierten Aktivität des Bi-213-d9MAk in verschiedenen Peritonealkarzinosestadien erfasst. Die mittlere Überlebenszeit von Tieren, die intraperitoneal mit 7,4 MBq Bi-213MAk am Tag 1 nach Tumorzellinokulation therapiert wurden, war mit 232 Tagen etwa zehnmal länger als jene von untherapierten Kontrolltieren, die bereits 24 Tage nach Tumorzellinokulation Aszites, große Tumormassen oder Tumokachexie zeigten. Bei spezifischer Therapie am Tag 8 nach Tumorzellinokulation, also bei fortgeschrittenem Karzinosestadium, verlängerte sich das Überleben der Mäuse auf 187 Tage. Der Unterschied im mittleren Überleben der Tiere nach Applikation von spezifischem Bi-213-d9MAk bzw. von unspezifischem Radioimmunkonjugat war nur bei Therapie am Tag 8 nach Tumorzellinokulation signifikant, da auch unspezifische Radioimmunkonjugate injiziert am Tag 1 nach Tumorzellinokulation deutliche Therapieerfolge bewirkten. Eine i.p. applizierte Aktivität von 22,2 MBq (600 µCi) führte etwa 180 Tage p.i. zu chronischem Nierenversagen. Ohne Nierenprotektion durch kationische Aminosäuren stellt die Niere somit das dosislimitierende Organ bei der Therapie mit Bi-213-d9MAk dar. Die Bewertung von Veränderungen der Tiergewichte ergab keinen toxischen Einfluss von 7,4 MBq (200 µCi) Bi-213-d9MAk auf den Säugetierorganismus. Die Leukozytenzahlen zeigten eine Abnahme in Abhängigkeit von der applizierten Aktivität, erholten sich aber innerhalb von 30 Tagen. Die Häufigkeit chromosomaler Aberrationen erhöhte sich mit zunehmender Aktivität bis 24 h nach Injektion des Bi-213-d9MAk. Zu späteren Zeitpunkten waren keine chromosomalen Aberrationen mehr nachweisbar. Das Radioimmunkonjugat Bi-213-d9MAk eignet sich nach den Resultaten dieser Studie hervorragend für die lokoregionale Anwendung bei disseminierter Tumorzellaussaat im Abdominalraum als Folge primären Magenkarzinoms mit Expression der d9-Mutante des E-Cadherins. Hohe therapeutische Effizienz, insbesondere bei Vorliegen von kleinen Tumorzellaggregaten und Einzelzellen, und niedrige Toxizität zeichnen das neue Radiopharmakon aus. Der therapeutische Einsatz des Bi-213-d9MAk empfiehlt sich besonders perioperativ bei Resektion eines soliden Magenkarzinoms zur Prophylaxe der Peritonealkarzinose aufgrund traumatischer Tumorzelldissemination.

31 histological eye specimens from dogs, cats and rabbits with secondary intraocular (metastatic) uveal tumours will be subject to a histological revision. These 31 specimens have been taken out of an overall pool of 3342 histological eye specimens, which were sent in to GSF Institute of Pathology between 1974 - 2003.