Tierärztliche Fakultät - Digitale Hochschulschriften der LMU - Teil 03/07 Archiv

Investigations into the diagnosis of the polyserositis complex in swine The aim of this study was to evaluate the PCR test results from collective swab samples of serous membranes for genomic fragments of H. parasuis and M. hyorhinis regarding their associations and correlations with the pretreatment, clinical and pathological findings as well as the detection of PRRSV EU strain and PCV-2. For the classification of the method in reference to H. parasuis the PCR results were compared with the results of the bacteriological examination of the BALF. Furthermore the association between PRRSV and PCV-2 as well as the correlations between the weight of the animals and the detection of the agents, the clinical score and the detection of the agents and between PCV-2 and H. parasuis, M. hyorhinis as well as PRRSV EU strain were determined. After the admission of the preliminary report, in which particularly the state of treatment and vaccination regarding H. parasuis was inquired, 143 pigs were clinically examined. The results were evaluated on the basis of a score system. From 117 pigs under neuroleptanalgesia bronchoalveolar lavage fluid was taken and examined bacteriologically for H. parasuis. The animals were euthanized afterwards and subjected to a gross pathological and histopathological examination. In the course of the necropsy collective swabs of the serous membranes were taken from the surfaces of pleura, pericard and peritoneum and analysed via PCR for genomic fragments of H. parasuis and M. hyorhinis. Additionally lung tissue samples were taken from 102 animals for the molecular biological proof of genomic fragments and tissue samples from lungs and lymph nodes were taken from 105 animals for in-situ hybridisation. The results show that the detection of the H. parasuis and M. hyorhinis in the PCR was not associated significantly with a previous treatment. No significant relation was found between the analysing methods, PCR from the collective serosal swab and bacteriological examination of the BALF. The detection of genomic fragments of H. parasuis showed a significant association with the findings of the auscultation. Furthermore the evidence of the agent in the PCR significantly correlated with the presence of CNS symptoms. The evaluation of the state of nutrition and the filling degree of the tarsal joints did not correlate significantly with the PCR proof of H. parasuis. In contrast significant relations were shown between the proof of genomic fragments of M. hyorhinis and the occurence of kyphosis, the findings of the auscultation and the evaluation of the state of nutrition, but the proof of genomic fragments of M. hyorhinis did not significantly correlate with the filling degree of the tarsal joints as well as the presence of CNS symptoms. The results of the pathological investigation showed a significant association of M. hyorhinis with the diagnosis of a catarrhal-suppurative bronchopneumonia as well as a significant association of both agents with the diagnosis of a serositis without differenciation of the site and for the singular diagnosis of a pleuritis and pericarditis. The proof of M. hyorhinis and H. parasuis in the PCR was not significantly associated with peritonitis and additionally that of H. parasuis was not significantly associated with the diagnosis of a catarrhal-suppurative bronchopneumonia. Significant associations were calculated between the detection of the following agents: H. parasuis and M. hyorhinis, H. parasuis and PRRSV EU strain as well as M. hyorhinis and PRRSV EU strain. In contrast the association between the detection of H. parasuis and PCV-2, M. hyorhinis and PCV-2 as well as PRRSV EU strain and PCV-2 was not significant. Furthermore associations were significant for the weight of the animals and the proof of PCV-2 respectively PRRSV EU strain, for the clinical score und the proof of H. parasuis respectively PCV-2 as well as for PCV-2 und PRRSV EU strain. In conclusion the sampling by collective dry swabs of the serous skins and the following PCR examination for genomic fragments is a meaningful method for the diagnosis of diseases, which are accompanied by polyserositis. With this procedure the proof of the agents can be carried out successfully in animals with clinical signs as well as in animals which are already pretreated with antibiotics. For H. parasuis and M. hyorhinis, both causal agents of polyserositis, a significant association was calculated. Both agents are capable of causing clinical signs and pathological findings associated with polyserositis. The disease induced by H. parasuis is called Glaesser´s disease, in reference to M. hyorhinis as Mycoplasma Polyserositis. Due to the existing association of both agents and the accompanied clinical and pathological signs it is reasonable to use the term “Polyserositis Complex”.

Investigations about the isoflurane anaesthesia in swine Compared to other animal species, swine show typical defensive behaviour towards any kind of physical manipulation. Anaesthesia is thus not only indicated for surgical interventions but for diagnostic and therapeutic measures as well. This study evaluates whether inhalational anaesthesia with isoflurane via facemask can serve as a potential alternative to regular intramuscular or intravenous anaesthesia. Two different apparati are compared: the first one disperses medical oxygen (O2), the second filtered compartment air (CA), both in conjunction with isoflurane. Animals with a body weight (BW) up to 20 kg receive simply an inhalational anaesthesia, patients weighing more than 20 kg in addition to that an intramuscular pre-medication 10 minutes earlier with ketamine (10 mg/kg BW) and azaperone (2 mg/kg BW). The survey examines 156 normal pigs and 19 statistically non-evaluated miniature pigs. Animals with more than 10 kg BW in particular show vehement physical reactions to the basal anaesthesia. Pre-medicated test animals are immobilized after ten minutes and the anaesthesia can be deepened with isoflurane. Animals receiving pure inhalational anaesthesia show ataxia after 29 to 34 seconds. After 48 to 60 seconds the probands are in prone position and after 65 to 89 seconds in lateral position. The pre-medicated animals on the other hand are significantly slower to show ataxia (after 1,4 minutes), prone position (after 1,8 to 1,9 minutes) and lateral position (4,3 to 4,4 minutes). The hypnosis and surgical tolerance stage of anaesthesia are reached significantly faster with isoflurane in CA than with isoflurane in O2. Test animals with isoflurane/O2 require an isoflurane-vaporiser position of 2,24 to 3,38 Vol% during the whole intervention, those with isoflurane/CA 2,17 to 2,88 Vol%. Compared to that, the premedicated animals need significantly less isoflurane with 1,5 to 2,38 Vol% (O2) and 1,52 to 2,08 Vol% (CA). Probands with O2 as carrier gas show a significantly better oxygen saturation (94-95%) than test subjects with CA (78%). The measured arterial pH value of 7,27 to 7,34 indicates a low acidosis; a partial pressure of carbon dioxide of 49,7 to 65,7 mmHg exceeds the reference values. The partial oxygen pressure is significantly better with O2 (190,0 to 266,2 mmHg) than with CA (57,4 to 65,7 mmHg). During the recovery phase, all animals exhibit the reappearance of the claw reflex, the dewclaw reflex, the muscle tension and positive skin sensibility in consecutive order. Somewhat later they raise their heads, get into the prone position and try to rise. Given several more minutes, they manage to stand. Probands receiving pure inhalation anaesthesia are significantly faster (1,3 to 9,8 minutes) to reach the waking stage than animals with a combination of ketamine, azaperone and isoflurane (2,1 to 24,4 minutes). The average blood pressure during the basal narcosis is 128/71 mmHg (systole/diastole) and 130/72 mmHg during the maintenance stage. Body weight and premedication apparently play no significant role. Miniature pigs deliver similar results as the premedicated pigs, but need remarkably less isoflurane (1,10 to 1,58 Vol%). Conclusion: a pure inhalation anaesthesia with isoflurane in swine can only be recommended for animals with a body weight up to 10 kg (O2-apparatus: 2,24 to 3,38 Vol% for surgical tolerance, CA-apparatus: 2,17 to 2,88 Vol% for surgical tolerance); rapid drifting off and rapid waking are the obvious advantages. A premedication with ketamine (10 mg/kg BW) and azaperone (2mg/kg BW) reduces the dose of isoflurane significantly (O2-apparatus: 1,5 to 2,38 Vol%, CA-apparatus: 1,52 to 2,08 Vol%). The use of O2 as carrier gas allows an optimal anaesthesia with optimal O2 - saturation for every animal weight and size (94 to 95%). CA as carrier gas can cause hypoxia and acidosis (O2-saturation: 78%, pH: 7,31 to 7,33, pCO2: 53,0 to 60,4 mmHg, pO2: 57,4 to 65,7 mmHg) during longer interventions. Miniature pigs need considerably less isoflurane, though this cannot result solely from the pre-medication and needs to be examined further.

Investigation of the distribution of Mycoplasma suis and comparing investigations of real-time PCR and acridine orange-stained blood smears concerning sensitivity and specificity As the porcine Eperythrozoonosis is most commonly a latent chronic infection, a high sensitive assay is required to evaluate the distribution of Mycoplasma suis in Germany. Due to its high sensitivity and specificity the real-time PCR was chosen for this study. In comparison to conventional PCR assays the real-time PCR is a rapid, easy-doing, more accurate and more sensitive system. In the present study the prevalence of M. suis in Germany was measured by using the LightCycler System and a constant real-time PCR-protocol. Additionally a new method of DNA-Extraction, the Gen Elute Bacterial Genomic DNA Kit, was evaluated during this study. The Gen Elute Bacterial Genomic DNA Kit showed the same results concerning sensitivity and efficiency of DNA extraction as the automatical extraction with the MagNA Pure LCTM System. 1176 blood samples of slaughtered post-weaning pigs from 196 different pig herds were collected. In addition to PCR analysis a haemogram was made from any sample and clinicochemical parameter were determined. Moreover an acridine orange-stained blood smear was analysed from each sample. Real-time PCR showed a positive result for 164 out of 1176 samples (13,9%). With the acridine stain only 35 pigs were identified as infected with M. suis. 31 of these samples were also positive in the real-time PCR. In the present study microscopic examination on stained blood smears only detected M. suis infections with a bacterial load of at least 105 per ml blood. 80 (40,8%) out of 196 pig herds were detected positive for M .suis by real-time PCR. The number of M. suis infected herds in the different districts of Germany varied from 33,3% to 48%. The prevalence within one herd varried from 25% to 46,2% and showed an average value of 34,2%. A significant correlation between the bacterial load per ml blood and the degree of severity of anemia was shown. A decrease of the number of erythrocytes, hemoglobin concentration and hematocrit was observed when the bacterial load increased. By comparing real-time PCR and microscopic examination of acridine orange-stained blood smears it was shown that the real-time PCR system is able to detect even latent M. suis infections that are missed out in the microscopic examination. Furthermore immature erythrocytes and Howell-Jolly-bodies may lead to false positive results. With the use of the M. suis specific hybridisation probe system in the real-time PCR false positive results can be avoided. The LightCycler MSG1 protocol has proven to be a high sensitive and easy-doing system that allows the integration in routine laboratories.

Investigation about the use of analgesics for the reduction of castration-induced pain in suckling piglets. This study is intended to investigate the implementation of analgesics in order to reduce pain induced by the castration of suckling piglets. Serum Cortisol and postoperative behaviour are used as parameters. Additionally, the influence on the healing process of the castration wounds is evaluated. 320 four to six day old male piglets with a good general condition and a birth weight over 1000g, are randomly allocated to group 1 to 15. The injections for the animals of group 12 to 15 are blinded. group number of animals application of the agent castration Cortisol 1 Handling NaCl 35 0,3 ml Saline Solution (0,9%) i.m. no 2 Handling Meloxicam 25 0,4 mg Meloxicam/kg wt i.m. no 3 Handling Flunixin 26 2,2 mg Flunixin-Meglumin/kg wt i.m. no 4 Handling Detomidin 25 40 μg Detomidinhydrochlorid/kg wt i.m. no 5 Castration NaCl 28 0,3 ml Saline Solution (0,9%) i.m. yes 6 Castration Meloxicam 25 0,4 mg Meloxicam/kg wt i.m. yes 7 Castration Flunixin 26 2,2 mg Flunixin-Meglumin/kg wt i.m. yes 8 Castration Metamizol + Skopolamin 25 50 mg Metamizol and 0,4 mg N-Butylscopolaminiumbromid/kg wt i.m. yes 9 Castration Metamizol 25 50 mg Metamizol/kg wt i.m. yes 10 Castration Carprofen 15 1,4 mg Carprofen/kg wt s.c. yes 11 Castration Detomidin 25 40 μg Detomidinhydrochlorid/kg wt i.m. yes Behaviour 12 Handling NaCl 10 0,3 ml Saline Solution (0,9%) i.m. no 13 Castration NaCl 10 0,3 ml Saline Solution (0,9%) i.m. yes 14 Castration Meloxicam 10 0,4 mg Meloxicam/kg wt i.m. yes 15 Castration Flunixin 10 2,2 mg Flunixin-Meglumin/kg wt i.m. yes 15 to 30 minutes after administering the substances, piglets are either castrated (groups 5-11 and 13-15) or only restrained for about 30 seconds (groups 1-4 and 12). They are returned to their box immediately afterwards. 2-3 ml blood is taken from all animals of the cortisol-groups before the application of the agents including 30 minutes, one hour, four and 24 hours after castration/fixation, and the concentration of cortisol in the serum is measured. The behaviour of all animals in groups 12 to 15 is observed individually for 20 minutes in the first and third hour after castration. Additionally, all piglets are scanned every 60 seconds for certain behaviours. On the first, fourth, seventh and 14th day after castration, wound healing and general condition are assessed and a “clinical score” is determined. The results of the cortisol measurement show that handling the animals has little influence on the cortisol level, while castration leads to a significant rise of the cortisol concentration that is still present four hours later. All of the tested non-opioid analgesics reduce the rise of the cortisol concentration after castration and a considerable reduction is especially noticeable after one hour. Group 6 “Castration Meloxicam” and group 7 “Castration Flunixin” show the lowest cortisol response to castration. The concentrations of cortisol in those two groups are significantly lower 30 minutes, one and four hours after castration than the concentration of group 5 “Castration NaCl”, and already after one hour they do not differ significantly from the corresponding handling groups. Irregardless if animals are castrated or only restrained, the use of Detomidin (group 4 and 11) leads to higher values of cortisol after castration/fixation compared to the values after the application of NaCl (group 1 and 5). A conspicuous impact on the postoperative behaviour is obvious when non-opioid analgesics are used. The frequency of occurrence of castration-induced signs of pain, drooping the tail and changing the position is explicitly reduced when Meloxicam and Flunixin are injected before castration. The effect of Flunixin is most clear. In this group, the incidence of castration-induced signs is only higher for five minutes after castration when compared to the Handling-group. Animals without preoperative medication show an overall reduced activity at the mammary glands and playing. Results indicate that non-opioid analgesics, especially efficient anti-inflammatory drugs like Meloxicam and Flunixin, are capable of reducing castration-induced pain in piglets. The castration wounds of 76,5% of the piglets are healed completely after 14 days. NSAIDs are incapable of improving the healing process.

Unmethylierte bakterielle DNA, die reich an CG Sequenzen ist stimuliert das angeborene Immunsystem. Die Vermittlung dieser Wirkung erfolgt über den Toll-like Rezeptor 9. Reine CG Sequenzen sind im Organismus nicht stabil, da sie sehr rasch von den körpereigenen Nukleasen abgebaut werden. Mit synthetisch hergestellten Oligodeoxyribonukleotiden, sogenannten CpG-ODNs, kann eine Nukleaseresistenz erzielt werden und der immunstimulatorische Effekt nachgeahmt werden. Weiterhin wirken sich CpG-ODNs positiv auf den Verlauf von bakteriellen Infektionen, Tumoren und Prioninfektionen aus. Man weiß, dass eine Aktivierung des Immunsystems durch einmalige Gabe von CpG-ODNs nur über einen kurzen Zeitraum stattfindet. Die CpG-ODNs führen im Organismus innerhalb von Minuten zu einem mRNA Anstieg und innerhalb von Stunden zu einer kurzfristigen Zytokinsekretion und IgM Produktion, werden dann aber rasch abgebaut, so dass der immunstimulatorische Effekt in der Regel nur kurz anhält. Um CpG-ODN als Therapeutikum besser nutzen zu können liegt der Wunsch nahe, die Wirkung von CpG-ODN zu verlängern. Eine mögliche Strategie ist hierbei eine repetitive Applikation. Ziel dieser Arbeit war, den immunstimulatorischen Effekt von repetitiver CpG-ODN-Gabe besser zu verstehen und mit der Repetition die Wirkung zu verlängern. Damit könnte CpG-ODN als Therapeutikum bei bakteriellen Infektionen, Tumoren und bei Prionerkrankungen wirkungsvoll eingesetzt werden. In dieser Arbeit wurden jeweils 12 Gruppen zu je 5 Mäusen gebildet, wobei die jeweiligen Gruppen an 5, 7 oder 21 aufeinanderfolgenden Tagen jeweils eine CpG-ODN, NaCl oder Neg-ODN Applikation i.p. erhielten. Die Mäuse wurden anschließend an Tag 7 oder 28 während bzw. nach der Behandlung getötet. So erhielt man folgende Gruppen: 5x-CpG-ODN, NaCl oder Neg-ODN behandelte, an Tag 7 getötete Mäuse 5x-CpG-ODN, NaCl oder Neg-ODN behandelte, an Tag 28 getötete Mäuse 7x-CpG-ODN, NaCl oder Neg-ODN behandelte, an Tag 7 getötete Mäuse 21x-CpG-ODN, NaCl oder Neg-ODN behandelte, an Tag 28 getötete Mäuse Im Verlauf der Arbeit, die die immunmodulatorische Wirkung auf das Gehirn und die Leber der Maus untersucht, konnten zusammenfassend folgende Ergebnisse herausgearbeitet werden, die mittels Real time PCR und Immunhistologie gewonnen wurden. Im Gehirn führt CpG-ODN zu einer mindestens einwöchigen Hochregulierung der mRNA von TNFα. Des Weiteren kommt es zu einer mindestens zweitägigen C1q und IFNg Hochregulierung. Eine IL-12p40 Hochregulierung findet nur ca. 18 Stunden statt, während eine STAT3 Hochregulierung nicht nachweisbar ist. In der histologischen Betrachtung finden sich in der HE Färbung keine pathologischen Veränderungen der Hirnarchitektur und in einer immunhistologischen Färbung mit CD 11b und GFAP bindenden Antikörpern keine Unterschiede zwischen den CpG-ODN, NaCl oder Neg-ODN behandelten Tieren. In der Leber findet sich eine signifikante Hochregulierung von IL-12p40 über mindestens drei Wochen und eine C1q, TNFα und IFNg Hochregulierung von mindestens einer Woche. Histologisch finden sich in der HE Färbung massive Leukozyteninfiltrate über mindestens zwei Tage. In der Immunhistologie sieht man an den Infiltraten beteiligte aktivierte Makrophagen, T und B-Lymphozyten, jeweils dargestellt mit CD 11b, CD 8 und B220 bindenden Antikörpern. Es zeigte sich, dass eine repetitive CpG-ODN-Gabe eine verlängerte stimulatorische Wirkung auf das Immunsystem ausübt, was Voraussetzung für den Einsatz als Therapeutikum ist. Besonders bemerkenswert ist die Tatsache, dass man durch periphere Gabe von CpG-ODN im Gehirn eine Immunstimulierung erreichen kann. Diese könnte im Rahmen einer Therapie von Prionerkrankungen, anderen Gehirninfektionen, Morbus Alzheimer oder Gehirntumoren Anwendung finden. Allerdings sind noch weitere Studien nötig, um Risiken wie Hepatotoxizität oder Autoimmunität besser abschätzen zu können und den Mechanismus zu erforschen, wie durch periphere Gabe von CpG-ODN eine immunologische Gehirnaktivierung erreichen wird. Eine wichtige Frage für die Zukunft ist hierbei, ob die Wirkung von CpG-ODN direkt auf die Gehirnzellen wirkt oder ob es einen second messenger gibt, der die Blut-Hirn-Schranke überwindet.