Im Graduiertenkolleg „Schlachttierbelastung und Produktsicherheit“ der Veterinärmedizinischen Fakultät der Universität Leipzig sollten in interdisziplinärer Zusammenarbeit Erkenntnisse zum Verhalten transportbelasteter Schlachtschweine in Bezug auf bakterielle Translokationsprozesse erarbeitet werden. Es wurden Mastschweine aus herkömmlichen Mastbetrieben definierten Belastungssituationen ausgesetzt , geschlachtet und untersucht. Dabei sollten physiologische, pathologisch-anatomische, immunologische, lebensmittel- und fleischhygienische, bakteriologische, virologische sowie ethologische Fragestellungen bearbeitet werden. Ziel war es festzustellen, ob eine Belastung der Tiere (u.a. durch den Transport) Auswirkungen auf die Produktqualität hat und ob durch eine Translokation pathogener Erreger ein Risiko für die Gesundheit des Verbrauchers besteht. Im Teilprojekt des Institutes für Virologie wurde untersucht, ob Rotavirus-Infektionen von Schlachtschweinen unter der Problematik der Belastung ein mögliches Infektionsrisiko für den Menschen darstellen. Um die zu erwartende niedrige Konzentration von Rotaviren in Organen von Schlachtschweinen nachweisen zu können, wurde eine kompetitive RT-PCR zum Nachweis von Rotaviren der Gruppe A verschiedener Spezies entwickelt. Dazu wurde ein sogenannter Kompetitor synthetisch hergestellt, welcher als interne oder externe Reaktionskontrolle eingesetzt wurde. Zum einen diente er der Überprüfung des ordnungsgemäßen Verlaufes einer RT-PCR, zum anderen wurde er zur Herstellung von Standards verwendet. Die RT-PCR wurde anschließend in eine „Real time“ RT-PCR umgewandelt. Sowohl mit der herkömmlichen als auch mit der „Real-time“ RT-PCR konnten 10 spezifische RNA-Moleküle in einer Probe nachgewiesen werden. In einer SPF-Schweineherde, welche einer Belastung infolge eines Tiertransports ausgesetzt war, konnten mit Hilfe der RT-PCR klinisch gesunde intermittierende Rotavirus-Ausscheider entdeckt werden. Bei einigen dieser Tiere gelang der Nachweis der Virusausscheidung über einen Zeitraum von drei Monaten. Nach der Schlachtung wurden in Organen des lymphatischen Systems bei zwei Schweinen sehr geringe Konzentrationen an rotavirus-spezifischer RNA detektiert. Infektiöses Virus konnte daraus allerdings nicht isoliert werden. Auch in einer Mastschweineherde konnte bei einigen Tieren Rotavirus-spezifische RNA im Kot nachgewiesen werden. Ein Infektionsversuch dieser Tiere mit Salmonella typhimurium konnte keine Reaktivierung der Rotavirus-Infektion auslösen. Aufgrund des Zoonose-Potentials von Rotaviren kann nach den Untersuchungen ein Infektionsrisiko für den Verbraucher durch eine endogene Kontamination von Schlachttieren mit Rotaviren nicht sicher ausgeschlossen werden. Die Untersuchungen zeigten auch, dass intermittierende Rotavirus-Ausscheider ein Infektionsrisiko für den Verbraucher darstellen, wenn z.B. bei der Schlachtung der Tiere oder bei der Verarbeitung des Fleisches dieser Tiere hygienische Grundregeln verletzt werden. Besonders gefährdet wären hierbei Neugeborene, Kinder, Senioren und immunsupprimierte Personen. Ein Ort einer Viruspersistenz in Organen konnte auch nach diesen Untersuchungen nicht gefunden werden. Dennoch scheint es, dass Rotaviren in der Natur oder in einer Population von Menschen oder Tieren persistieren. Durch ständige Neuinfektionen bzw. Reinfektionen empfänglicher Organismen haben Rotaviren so ihre Erhaltung gesichert. / Within the graduate programme “Schlachttierbelastung und Produktsicherheit” of the Veterinary Faculty of the University of Leipzig, the behaviour of slaughter swine exposed to the stress of transport was observed in an interdisciplinary collaboration concerning the translocation processes of bacteria. Fattened pigs from conventional pig fattening units were exposed to particular stress situations, and then slaughtered and examined. The following aspects of this process were investigated: physiology, pathological-anatomy, immunology, food and meat hygiene, bacteriology, virology and ethology. The aim of this study was to verify whether exposing the animals to stress situations (such as transport) influences the quality of the product and whether the translocation of pathogens represents a risk for consumer health. Within the sub-project of the Institute for Virology, analyses were made to verify whether rotavirus infections of slaughter swine exposed to stress situations represents a potential contamination risk for humans. In order to detect the expected low concentration of rotaviruses in the organs of slaughtered pigs, a competitive RT-PCR was developed as a test of rotaviruses for various group A species. To do this, a so-called competitor was synthetically created, which was used as an internal and external reaction control. On one hand, it was used to verify the regular course of an RT-PCR reaction, and on the other hand, it was implemented to develop standards. RT-PCR was then modified by means of a real time RT-PCR. Both with the conventional and with the real time RT-PCR, it was possible to detect 10 specific RNA molecules/ sample.With this new very sensitive and specific amplification process, it was possible to detect rotavirus-specific RNA in the excrement of people and of pigs, cows, horses, rabbits and monkeys. the evidence of the virus excretion was produced over a time period of three months. After slaughtering, low amounts of rotavirus specific RNA were found in the organs of the lymphatic system. There were no indications that any of these organs were infectious. Rotavirus specific RNA was also found in the excrement of some fattened pigs. An attempt to infect these animals with Salmonella typhimurium was unable to cause any reactivation of the rotavirus infection. An infection risk for the consumer through an endogenous rotavirus contamination of fattened pigs cannot be excluded with any degree of certainty on the basis of these analyses due to the zoonotic potential of rotaviruses. The analyses also showed that intermittent rotavirus excretors represent an infection risk for the consumer, if for example basic hygiene rules are broken during the slaughter or meat processing of these animals. At special risk may be new-borns, children, youth, the elderly and people suffering from immunodeficiency. These examinations could not find a specific place in the organs where the virus persists. Nevertheless, it seems that rotaviruses persist in the environment or in a population of people or animals. With constant new infections or re-infections of receptive organisms, rotaviruses seem to have assured their survival.

Polymerase Chain Reaction (PCR) detection paves the way to reliable and rapid diagnosis of diseases and has been used extensively since its introduction. Many miniaturized PCR systems were presented by microfluidics and lab-on-a-chip community. However, most of the developed systems did not employ real-time detection and thus required post-PCR processes to obtain results. Among the few real-time PCR systems, almost all of them aimed for medical applications and those for plant pathogen diagnosis systems are almost non-existent in the literature. In this work, we are presenting a portable system that employs microfluidics PCR system with integrated optical systems to accomplish real-time quantitative PCR for plant pathogen diagnosis. The system is comprised of a PCR chip that has a chamber for PCR sample with integrated metal heaters fabricated by standard microfabrication procedures, an optical system that includes lenses, filters, a dichroic mirror and a photomultiplier tube (PMT) to achieve sensitive fluorescence measurement capability and a computer control system for Proportional Integral Derivative (PID) control and data acquisition. The optical detection system employs portable components and has a size of 3.9 x 5.9 x 11.9 cm which makes it possible to be used in field settings. On the device side, two different designs are used. The first design includes a single chamber in a 25.4 x 25.4 mm device and the capacity of the chamber is 9 micro-liters which is sufficient to do gel electrophoresis verification. The second design has three 2.2 micro-liter chambers squeezed in the same size device while having smaller volume to increase high throughput of the system. The operation of the system was demonstrated using Fusarium oxysporum spf. lycopersici which is a fungal plant pathogen that affects crops in the USA. In the presence of the plant pathogen, noticeable increases in the photomultiplier tube output were observed which means successful amplifications and detections occurred. The results were confirmed using gel electrophoresis which is a conventional post-PCR process to determine the existence and length of the amplified DNA. Clear bands located in the expected position were observed following the gel electrophoresis. Overall, we have presented a portable PCR system that has the capability of detecting plant pathogens.

To enable assessment of recombinant BFDV capsid protein (recBFDVcap) for vaccination to protect against PBFD, commercially available lovebirds (Agapornis sp.) were tested for evidence of past and current BFDV infection using PCR, HI and HA to identify suitable BFDV-free birds in which to test the vaccine. During this attempt, it was found that lovebirds from commercial aviaries were endemically infected with BFDV with evidence of up to 100% prevalence of BFDV DNA in blood samples from individual birds over time. Such an approach was abandoned as unlikely to yield suitable numbers of naïve birds to conduct a BFDV vaccination trial. As commercially available lovebirds were considered to be a poor source of BFDV-free birds, wild caught cockatoo nestlings and eggs (long-billed corella; Cacatua tenuirostris and galah; Eolophus roseicapillus) were used to assess the efficacy of BFDV vaccination using baculovirus recombinant BFDV capsid. Eggs were artificially incubated and 3 eggs successfully hatched and 1 was successfully hand-reared. All nestlings were screened for BFDV DNA in blood using PCR upon arrival then on days 11, 18 and 25 and tested for anti-BFDV antibody on the day of arrival. All hatched birds were determined to be free of BFDV DNA and BFDV HI antibody in the peripheral blood throughout the hand rearing period and the flock was considered to be suitable for a BFDV vaccination trial. Corellas (n=13) were injected with 1 mL of vaccine containing 10 μg recBFDVcap on day 0 and 0.4 mL vaccine containing 66.8 μg recBFDVcap on day 11. All vaccinated corellas and 5 non-vaccinated control corellas were given 0.4 mL BFDV suspension (titre = log2 12 HAU/50 μL) intramuscularly and 0.1 mL orally 16 days after booster vaccination. Blood was collected periodically during the vaccination period and blood and feathers collected before and after BFDV administration. Testing included BFDV DNA detection by PCR and qRT PCR (on blood) as well as serum antibody detection by haemagglutination inhibition (HI) and BFDV DNA and antigen was detected by qRT PCR and haemagglutination (HA) (on feathers), respectively. Four of 97 blood samples collected from vaccinated birds post BFDV challenge tested positive by PCR, whereas 17 of 35 samples taken from non-vaccinated control corellas tested positive. Vaccinated birds did not develop feather lesions, had only transient PCR detectable viraemia and had no evidence of persistent infection 270 days post-challenge using PCR, histopathology and immunohistochemistry (IHC). Non-vaccinated control corellas developed transient feather lesions and PCR, HI and HA test results consistent with PBFD. They were BFDV PCR positive for up to 41 days post-challenge and qRT PCR demonstrated reduced virus replication in vaccinated birds compared to non-vaccinated control birds. Thus, administration of recBFDVcap vaccine alone was found to incite an adaptive immune response in BFDV-free corellas that subsequently conferred protection against inoculation with BFDV. A commonly utilized method for excising blood dried on filter paper was proven to be of high risk of carryover contamination facilitated by a hole punch used for processing several samples. Therefore a practical method of avoiding carryover contamination was developed and used in the DNA testing procedures of the vaccination trial. Finally, the haematological characteristics of the above mentioned cockatoos were studied before and for 97 days after experimental infection with BFDV. It was found that the pre-challenge haematological values were similar between the vaccinated and non-vaccinated corellas. Most pre-challenge parameters were comparable to previously reported values of other cockatoos and psittacine birds. Significant differences were seen in both groups when comparing pre-challenge values with post challenge values for total and differential leukocyte concentrations, but PCV and TSP were not significantly affected by BFDV challenge.

The exact role of mycoplasma in feline ocular and respiratory disease is not yet understood. The results of previous studies are contradictory in this regard. There is some evidence to suggest that M. felis has a pathogenic role in such diseases, but it is inconclusive. / The aim of this study was to investigate the prevalence and anatomical distribution of mycoplasmas in a population of shelter cats, to determine which species were present, and establish the association of their presence with ocular or respiratory disease. / The prevalence of mycoplasma in the 110 cats examined was 71.8%, as determined by in vitro culture. Mycoplasma was most commonly isolated from the pharynx, followed by the bronchus and conjunctiva. In infected cats, mycoplasmas were likely to be isolated from multiple anatomical sites. / The polymerase chain reaction (PCR) was used to amplify part of the 16S rRNA gene, and the mutation scanning technique non-isotopic single-strand conformation polymorphism (SSCP) was utilised to delineate mycoplasma isolates based on nucleotide sequence variation. PCR-SSCP proved to be a useful method to screen large numbers of samples for variation and to group them according to species. / The species of mycoplasma identified by nucleotide sequencing were M. felis and M. gateae. It was not determined whether it was possible to differentiate between M. gateae and M. arginini based on SSCP profile results with the target DNA region used due to their almost identical nucleotide sequence. This group of M. gateae/M. arginini served as a useful non-pathogenic comparison group to M. felis. / There was no statistically significant difference between M. felis and the M. gateae/M. arginini group with respect to prevalence or anatomic distribution. There was no evidence of any association of mycoplasma with disease linked to any of the anatomic locations studied. / Mycoplasmas were isolated from the lower respiratory tract in 42.7% of cats. The isolation of mycoplasmas from the lower respiratory tract of healthy cats has been reported once, but this is the first report of M. felis being isolated from this location in healthy cats. This finding indicates that the isolation of mycoplasmas from the lower respiratory tract is not sufficient evidence to implicate a role in respiratory disease. / Mycoplasmas were not significantly involved in ocular or respiratory disease in the population of cats studied. More likely, they are commensal organisms in the conjunctiva, pharynx and bronchus. Whether they are capable of playing an opportunistic role in disease, or what conditions may facilitate such a role remains to be determined.

The age structure of populations is an important and often unresolved factor in ecology and wildlife management. Parameters like onset of reproduction and senescence, reproductive success and survival rate are tightly correlated with age. Unfortunately, age information of wild animals is not easy to obtain, especially for birds, where few anatomical markers of age exist. Longitudinal age data from birds banded as chicks are rare, particularly in long lived species. Age estimation in such species would be extremely useful as their long life span typically indicates slow population growth and potentially the need for protection and conservation. Telomere length change has been suggested as a universal marker for ageing vertebrates and potentially other animals. This method, termed molecular ageing, is based on a shortening of telomeres with each cell division. In birds, the telomere length of erythrocytes has been reported to decline with age, as the founder cells (haematopoietic stem cells) divide to renew circulating red blood cells. I measured telomere length in kakapo, the world largest parrot and four other bird species (Buller’s albatross, kea, New Zealand robin and saddleback) using telomere restriction fragment analysis (TRF) to assess the potential for molecular ageing in these species. After providing an overview of methods to measure telomere length, I describe how one of them (TRF) measures telomere length by quantifying the size distribution of terminal restriction fragments using southern blot of in-gel hybridization (Chapter 2). Although TRF is currently the ‘gold standard’ to measure telomere length, it suffers from various technical problems that can compromise precision and accuracy of telomere length estimation. In addition, there are many variations of the protocol, complicating comparisons between publications. I focused on TRF analysis using a non-radioactive probe, because it does not require special precautions associated with handling and disposing of radioactive material and therefore is more suitable for ecology laboratories that typically do not have a strong molecular biology infrastructure. However, most of my findings can be applied to both, radioactive and nonradioactive TRF variants. I tested how sample storage, choice of restriction enzyme, gel Abstract II electrophoresis and choice of hybridization buffer can influence the results. Finally, I show how image analysis (e.g. background correction, gel calibration, formula to calculate telomere length and the analysis window) can not only change the magnitude of estimated telomere length, but also their correlation to each other. Based on these findings, I present and discuss an extensive list of methodological difficulties associated with TRF and present a protocol to obtain reliable and reproducible results. Using this optimized protocol, I then measured telomere length of 68 kakapo (Chapter 3). Almost half of the current kakapo population consists of birds that were captured as adults, hence only their minimum age is known (i.e. time from when they were found +5 years to reach adulthood). Although molecular ageing might not be able to predict chronological age accurately, as calibrated with minimum age of some birds, it should be able to compare relative age between birds. Recently, the oldest kakapo (Richard Henry) was found to show signs of reproductive senescence. The age (or telomere length) difference to Richard Henry could have been used to approximate the remaining reproductive time span for other birds. Unfortunately, there was no change of telomere length with age in cross sectional and longitudinal samples. Analysis of fitness data available for kakapo yielded correlations between telomere length and fledging success, but they were weak and disappeared when the most influential bird was excluded from analysis. The heavy management and small numbers of kakapo make conclusions about fitness and telomere length difficult and highly speculative. However, telomere length of mothers and their chicks were significantly correlated, a phenomena not previously observed in any bird. To test if the lack of telomere loss with age is specific to kakapo, I measured telomere length of one of its closest relatives: the kea (Chapter 4). Like kakapo, telomere length did not show any correlation with age. I then further assessed the usefulness of molecular ageing in birds using only chicks and very old birds to estimate the maximum TL range in an additional long lived (Buller’s albatross) and two shorter lived species (NZ robin and saddleback). In these Abstract III species, telomere length was on average higher in chicks than in adults. However, age matched individuals showed high variations in telomere length, such that age dependent and independent telomere length could not be distinguished. These data and published results from other bird species, coupled with the limitations of methodology I have identified (Chapter 2), indicate that molecular ageing does not work in most (if not all) birds in its current suggested form. Another way to measure telomere length is telomere Q-PCR, a real-time PCR based method. Measurement of the same kakapo samples with TRF and Q-PCR did not result in comparable results (Chapter 4). Through experimentation I found that differences in amplification efficiency between samples lead to unreliable estimation of telomere length using telomere Q-PCR. These differences were caused by inhibitors present in the samples. The problem of differential amplification efficiency in Q-PCR, while known, is largely ignored by the scientific community. Although some methods have been suggested to correct for differing efficiency, most of these introduce more error than they eliminate. I developed and applied an assay based on internal standard oligonucleotides that was able to corrected EDTA induced quantification errors of up to 70% with high precision and accuracy (Chapter 5). The method, however, failed when tested with other inhibitors commonly found in DNA samples extracted from blood (i.e. SDS, heparin, urea and FeCl3). PCR inhibition was highly selective in the probe-polymerase system I used, inhibiting amplification of genomic DNA, but not amplification of internal oligonucleotide or plasmid standards in the same reaction. Internal standards are a key feature of most diagnostic PCR assays to identify false negatives arising from amplification inhibition. The differential response to inhibition I identified greatly compromises the accuracy of these assays. Consequently, I strongly recommend that researchers using PCR assays with internal standards should verify that the target DNA and internal standard actually respond similarly to common inhibitors.

Neotyphodium endophytes (Claviceptaceae) are asexual filamentous fungi found living between the cells of many cool season forage grasses including tall fescue, meadow fescue and perennial ryegrass. They produce a range of alkaloids, including ergovaline and lolitrem B, which have been shown to be directly associated with the livestock disorders fescue toxicosis and ryegrass staggers syndrome, while others, including peramine and the lolines, have been linked to increased insect and drought resistance of the grass host. In the past decade, the Neotyphodium strains AR1, MaxQ and MaxP were selected because they did not produce the alkaloids associated with livestock disorders. Subsequently, artificial associations were established between them and commercial forage grass cultivars. The slow growth rate of Neotyphodium endophytes in vitro and the low success rate of the present methods for establishing artificial associations between endophytes and grass hosts are limiting the rate at which new novel endophytes can be incorporated into plant breeding programs and eventually commercialised. In this thesis, the type and concentration of the growth medium was shown to affect radial growth rate, colony appearance and mycelial morphology of three strains of Neotyphodium endophytes. The floret inoculation of meadow fescue with the U2 strain of N. uncinatum using several techniques involving liquid culture was attempted but was unsuccessful in creating any artificial associations. Neotyphodium endophytes are unstable in stored seed. In New Zealand, it is critical that pastures are infected with protective Neotyphodium endophytes to ensure that they will not be destroyed by exotic pests. The present methods for determining the percentage of viable endophyte infection of a seed lot are too slow for efficient use in the commercial seed industry. In this thesis, primers specific to the â-tubulin gene of N. coenophialum, N. lolii and N. uncinatum were designed and successfully used to detect these species in planta. However, using these primers to develop a method to accurately determine the viable endophyte infection rate of a seed lot using RT-qPCR was unsuccessful.

No description available.

Canine tick-borne diseases are an emerging problem within Australia and throughout the world. This thesis investigates Babesia gibsoni and Anaplasma platys infections in dogs in Australia and also explores the evolutionary relationships and taxonomy of the canine piroplasm species and the members of the order Piroplasmida. A nested PCR-RFLP assay was developed for the detection and differentiation of the canine piroplasm species and was found to have a high detection limit, capable of detecting a 2.7 x 10-7 % parasitaemia or the equivalent of 1.2 molecules of target DNA. Detection of piroplasm DNA applied to Whatman FTA“ classic cards using nested-PCR was found to have a lower detection limit than when using DNA extracted from whole blood but higher than IsoCode‘ Stix or QIAamp extraction from filter paper based techniques. The nested PCR-RFLP assay was further evaluated for the detection of B. gibsoni infection in dogs being exported from Australia to New Zealand and compared to the current screening methods, the Immunofluorescent Antibody Test (IFAT) and microscopy. Of 235 dogs screened, 11 were IFAT positive, 1 was microscopy positive and 3 were PCR positive for B. gibsoni, highlighting the discordance that exists between various detection techniques. Replacing microscopic examination of blood smears with PCR-RFLP is suggested for screening dogs entering New Zealand, in addition to revising the current IFAT cut-off titre to minimize false positive results. The first case of B. gibsoni in New South Wales is also reported. A study was also conducted to further investigate the recent discovery of B. gibsoni in Australia and the association of this infection with American Pit Bull Terriers in an epidemiological study. Both American Pit Bull Terriers (n = 100) and other dog breeds (n = 51) were screened for B. gibsoni using IFAT and PCR-RFLP. A questionnaire was also completed by each dog owner regarding thethe husbandry and habits these dogs. Fourteen dogs were positive for B. gibsoni using IFAT and/or PCR-RFLP and all were American Pit Bull Terriers. Dogs that were male and/or were bitten by or were biters of other American Pit Bull Terriers were statistically more likely to be B. gibsoni positive, thus suggesting that blood-to-blood transmission may contribute to the spread of this disease. Experimental B. gibsoni infections were established in vivo to investigate the efficacy of combined atovaquone and azithromycin therapy and to determine the detection limits of PCR, IFAT and microscopy during various stages of infection. While atovaquone and azithromycin produced a reduction in circulating parasite levels, it did not cause total eradication, and possible drug resistance also developed in one dog. PCR was found to be most useful in detecting early and acute stage infections, while IFAT was most useful during chronic and acute infections. Microscopy is suggested to be only useful for detecting acute stage infections. This study also describes the detection of B. gibsoni in tissue samples during chronic infection for the first time, suggesting possible sequestration of this parasite. Anaplasma platys has also only recently been reported in Australia and the distribution, molecular-charcterisation, pathogenesis, co-infection with Babesia canis vogeli and treatment of infection with doxycycline were investigated. For the first time, A. platys is reported in Western Australia, Queensland and Victoria, with each isolate found to be genetically identical on the basis of the 16S rRNA gene. No correlation could be established between A. platys infection and the development of clinical signs or pathogenesis and definitive treatment using doxycycline could not be determined. Isolates of canine piroplasms from various geographical locations worldwide (n = 46), including Australia were characterised on the basis of multiple gene loci to explore the distribution, genetic variation and possible phylogeographical relationships of these species. Separate genotypes of B. canis vogeli, B. canis canis and B. gibsoni are suggested and may be correlated to different geographical origins. Characterization of B. canis vogeli, B. canis canis and B. canis rossi on the basis of the HSP 70 gene and B. gibsoni on the basis of the ITS 1, 5.8S rRNA gene and ITS 2 is described for the first time. Elevation of each of the B. canis subspecies, with the exclusion of B. canis presentii, to separate species is also proposed. The current paraphyly and taxonomic confusion associated with the members of the order Piroplasmida led to a review of the phylogenetic and taxonomic status of this group of organisms. Phylogenetic relationships are determined using 18S rRNA gene, 5.8S rRNA gene, HSP 70 gene and combined loci analyses. Rearrangement of the Piroplasmida into three families, including the new family Piroplasmiidae is proposed, in addition to the establishment of two new genera, the Piroplasma (Patton, 1895) and the Achromaticus (Dionisi, 1899). Other proposed schemes of classification and the limitations of phenotypic characteristics in taxonomic classification within the Piroplasmida are also discussed.

Würzburg, Universiẗat, Diss., 2007.

Thesis (M. Sc.) Uppsala : Sveriges lantbruksuniv., 2006.