Tierärztliche Fakultät - Digitale Hochschulschriften der LMU - Teil 04/07 Archiv

The aim of this double-blinded, randomised and placebo-controlled study was to assess the potential influence of polyunsaturated fatty acids on the inflammatory infiltrate in the skin of twenty-eight mixed breed dogs diagnosed with atopic dermatitis. The animals ranged from 1 to 10 years of age. They were assigned to one of three groups and supplemented for ten weeks with flax seed oil, a commercial fatty acid product or a placebo. Skin punch biopsy specimens from the lateral thorax were taken prior to and at the end of the trial. Clinical signs as well as pruritus intensity were evaluated by owner pruritus scores and clinician lesion scores. The number of mast cells and eosinophils per mm2 within the epidermis and dermis were quantified before and after supplementation by utilisation of Image Analysis Software. There was no significant difference between cell counts before and after the study nor when comparing groups amongst themselves. However, a positive correlation between cell counts and the pruritus intensity as well as cell counts and clinical signs was observed.

The vaccination against avian paramyxovirus of the serotype 1 (APMV-1), the highly contagious agent of Newcastle Disease, is usually conducted with live attenuated vaccine strains within the first two weeks after hatch. Multiple efforts have been made to develop an in ovo vaccine, yet none of the approaches proved suitable for an application in commercial poultry. The aim of the performed study was to evaluate the strain APMV-1 599, which had proven apathogenic for day-old chicks, for its progress of infection and the induction of protective immunity after inoculation of spf-chicken eggs. The inoculation of embryonated chicken eggs with 106 EID50 of the APMV-1 599 strain at incubation day 18 caused an infection in virtually all hatched chicks at a maintained hatch rate of 81,7%, which was not substantially lower than that the 60% hatch rate of a group of uninfected sentinel animals that hatched in the same incubator and the hatch rate of 95% of an uninfected and separately hatched control group. Although the hatch rate was not reduced, the animals that were infected in ovo with APMV-1 599 showed pronounced clinical signs during the first ten days after hatch. These manifested themselves in the form of respiratory symptoms, apathy, diarrhea and in sporadic cases central nervous impairments, which led to a mortality rate of 54,1% within the first eight days after hatch. Opposed to this, the affiliated sentinel animals showed neither distinct clinical signs nor a substantially elevated mortality rate despite the proven transmission of the virus by the animals of the in ovo infected goup. To determine the spread of the virus to numerous organs, a novel Real-Time RT-PCR protocol targeting an amplificate located at the 5’ end of the nucleoprotein gene was established in addition to the published and validated method targeting a sequence on the matrix protein gene (WISE et al, 2004). Due to low fluorescence levels and irregular fluorescence curves, six of the conventional nucleotides incorporated into the 5’ nuclease probe were replaced by “locked nucleic acid” (LNA) nucleotides. This led to a shortening of the probe from 25 to 17 bases and thus an improvement of probe hybridization. The newly developed assay showed a comparable performance to the previously established protocol. During the first eight days after hatch, viral RNA was detectable in multiple organs with a predilection for lung and cecum / cecal tonsil. Viral RNA could also be detected in twelve of the liver samples as well as nine of the brain samples. Following the period of clinical impairment, no viral RNA was detectable in the samples from days 14 and 21. Specific antibodies could be detected by hemagglutination inhibition test from day seven and from day 21 by commercial ELISA. Additionally, the evaluation of cellular immune response indicates the formation of t-memory cells in the in ovo infected group and the sentinel animals. One single inoculation of APMV-1 599 in ovo was sufficient to induce a humoral and cellular immune response to protect all in ovo infected and most of the sentinel animals upon challenge with velogenic NDV Herts (Weybridge 33 / 56) at day 21. Shedding of the challenge virus was not noted in the in ovo infected group, while three of the ten sampled sentinel animals as well as all the animals in the control group shed virus two and four days after challenge, respectively. Due to the visible clinical impairment of the chicks, an in ovo vaccination with APMV-1 599 in the applied mode is not to be considered. The performed experiments demonstrated a generalization of the infection and servere clinical symptoms compared to the vaccination of animals after hatch. These results are indicative of embryonic developmental processes within the last three days before hatch, which are essential for the immunologic defense of very young chicks. A complete understanding of these ongoing immunological processes would not only be of importance for the development of in ovo vaccines against Newcastle Disease, but also in regard to potential early infections with other infectious agents.

The aim of the present study was to evaluate the relevance of an Avian Orthoreovirus (ARV) isolated from broiler chicken suffering from infectious runting and stunting syndrome (RSS) after simulated vertical infection of chicken. The influence of the age at infection on the progression of ARV infection is of particular importance. At first, the consequences of an early in ovo inoculation with the ARV-isolate 8474DP4-6 in specific pathogen free (spf) embryonated chicken eggs were evaluated. Infected embryos that survived until the 19th day of incubation were observed regularly after inoculation into the allantoic cavity of embryonated chicken eggs with varying doses of the ARV-isolate 8474DP4-6 on the ninth day of incubation. The inoculation into the yolk sac of embryonated chicken eggs on the sixth day of incubation with the same virus resulted in surviving infected embryos only in singular cases. On the other hand, infected embryos died after in ovo inoculation with the ARV vaccine strain S1133 into the allantoic cavity or the yolk sac on the corresponding days of hatch. The mean death time for the infected embryos displayed a high variability. The mean death time after in ovo inoculation with the ARV strain S1133 was observed between 100 and 168 hours while embryos infected with the ARV-isolate died between 60 and 120 hours post inoculation. For a subsequent animal experiment embryonated chicken eggs (Lohmann Selected White Leghorn) were inoculated with the ARV-strain 8474DP4-6 into the allantoic cavity on the 14th day of incubation and the hatched chickens were raised until the age of 36 days. Inoculated birds were kept together with non-inoculated sentinels while a mock-infected group was raised separately. The in ovo inoculation resulted in the hatch of ARV-infected chicks. The hatchability was reduced in the infected group (56%) as well as in the mock-infected control group (46%). ARV was reisolated regularly from organs of infected chicks until the 12th day post hatch and sporadically until day 36 post hatch. Several organs (liver, duodenum and pancreas [pooled], proventriculus, jejunoileum and bursa of Fabricius) were sampled in order to evaluate the organotropism and the progression of infection in inoculated chicks. ARV was reisolated from all kinds of examined organs, whereas the isolation from intestinal organs occurred more frequently. The transmission to non-inoculated sentinels was demonstrated by reisolation of ARV. The inoculated as well as the sentinel chicks developed antibodies against ARV. The results demonstrate the possibility of a vertical and likewise a horizontal transmission for the ARV-strain 8474DP4-6. An increased mortality became evident within the infected group. From the day of hatch and the seventh day of life 52,2% of the animals died, until the 12th day of life the mortality peaked to 56,5%. Within the control group a mortality of 6 and 12% respectively was observed during the corresponding period. Inoculated as well as control animals were of poor condition and suffered from diarrhea. Not inoculated control birds recovered quickly after an antibiotic treatment while the inoculated chicken did not respond well to the therapy. The gross pathology did not reveal any alterations in neither of the groups. Histopathological changes included myocarditis, pancreatic degeneration and lymphocyte depletion in the spleen. Within the bacteriological examination various ubiquitous bacterial species including Staphylococcus sp., Streptococcus sp., Enterococcus sp., Bacillus sp., Escherichia coli, Citrobacter freundii and Enterobacter cloacae were cultivated from organ swabs from animals of both groups. Body weight and weight gain were significantly decreased in inoculated chicks from day seven post hatch on. In ovo infection of embryonated eggs from ARV-vaccinated broiler breeders with the ARV-isolate 8474DP4-6 resulted in a hundredfold lower egg infectious dose 50 compared to embryonated spf chicken eggs. Furthermore, a larger proportion of infected embryos survived until the 19th day of incubation. In conclusion, the in ovo inoculation of embryonated eggs with the RSS-associated ARV strain resulted in the impairment of hatched chicks. The bacteriological findings indicate an increased susceptibility of the ARV-infected chicks to bacterial overgrowth. Future experiments evaluating the effects of early ARV-infections with the focus on alterations of the immune-response will help to elucidate the role of early ARV-infections in chicks.

The objective of this study was to assess the efficacy of Porcilis PCV administered as a single dose to piglets at either one or three weeks of age as compared to a placebo group that received Diluvac Forte twice by injection.

Ziel der vorliegenden Dissertationsarbeit war es, die mögliche Empfänglichkeit von Karpfen (Cyprinus carpio), Koi-Karpfen (Cyprinus carpio koi) und Zebrabärblingen (Danio rerio) gegenüber der Drehkrankheit mittels Nested-PCR und histologischen Untersuchungen zu klären. Zum Vergleich wurden Drehkrankheit-empfindliche Regenbogenforellen (Oncorhynchus mykiss) mit untersucht. Für die Untersuchungen zum Eintritt von Triactinomyxon-Sporen wurden Regenbogenforellen, Karpfen, Koi-Karpfen und Zebrabärblinge exponiert und jeweils 5 Minuten, 4, 6, 8, 12 und 18 Stunden, 1, 2, 3 und 5 Tage nach der Exposition die Parasitenstadien in der Schwanzflosse und in der Haut (an der lateralen Seite) der Forellen gezählt. Bei den Regenbogenforellen wurden in den Schwanzflossen nach fünf Minuten und vier Stunden Parasitenzahlen zwischen 16 bis 25 auf einer Fläche von 1 cm² pro Hautstück (an der lateralen Seite) gefunden, während bei allen drei untersuchten Cypriniden durchschnittlich 5 intakte eingedrungene Amöboidkeimzellen pro Fisch histologisch nachweisbar waren. In der Schwanzflosse und in der Haut von Karpfen, Zebrabärblingen, Koi-Karpfen und Regenbogenforellen war zu den Untersuchungszeitpunkten 1, 4 und 8 Stunden p.e. DNA von M. cerebralis nachweisbar. Nach acht Stunden waren bei den Regenbogenforellen und bei allen drei untersuchten Cypriniden die Ambödkeimzellen noch intakt. Außerdem war eine Abnahme der Anzahl an Parasitenstadien in der Haut nur bei den Regenbogenforellen zu detektieren. Ab 8 Stunden bis 60 Tage post expositionem konnte bei Regenbogenforellen in den Schwanzflossen ein Rückgang der Infektionsintensität festgestellt werden. In der Epidermis der Schwanzflossen war die durchschnittliche Triactinomyxon-Sporen-Dichte mit Abstand am höchsten. Es konnte jedoch zum Untersuchungszeitpunkt 60 Tage bei Regenbogenforellen Parasiten-DNA mittels Nested-PCR nachgewiesen werden, obwohl diese Fische bei der histologischen Untersuchung negativ getestet wurden. Ab 50 Tage post expositionem waren nur bei den Regenbogenforellen im Kopfknorpel Entwicklungsstadien von M. cerebralis und Veränderungen in Form von Degenerationen zu finden. Alle drei untersuchten Cypriniden wiesen im Kopfknorpel keine Entwicklungsstadien von M. cerebralis oder Schädigungen auf. Nach den Ergebnissen der vorliegenden Arbeit kann somit vermutet werden, dass viele Sporoplasmen in die Epidermis der Cypriniden eindrangen, aber den Knorpel nicht erreichen konnten. Der Beweis für die erfolgte Infektion des Schädelknorpels der Regenbogenforellen mit M. cerebralis wurde anhand der klinischen und histologischen Befunde durchgeführt, während in allen drei untersuchten Cyprinidenarten, der Parasit die Haut eindrangenx , aber nicht weiter entwickeln konnte. Die aus Hautproben, Schwanzflossen und Knorpelproben der infizierten Karpfen, Koi-Karpfen, Zebrabärblinge und Regenbogenforellen gewonnene DNA wurde mittels Nested-PCR amplifiziert. Dabei stellte sich heraus, dass die Nested-PCR deutlich sensitiver war als die histologische Untersuchung mit Hämatoxylin- und Eosin- Färbung. Aufgrund der vorliegenden Ergebnisse können weiterführende Untersuchungen durchgeführt werden, um die Resistenzmechanismen der Cypriniden gegenüber der Drehkrankheit klären zu können.