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Mycoplasma hyopneumoniae is het primaire agens van enzoötische pneumonie en speelt een belangrijke rol in het porcien respiratoir ziektecomplex. In de meeste landen is de kiem endemisch aanwezig en varkens van alle leeftijden kunnen besmet worden. De meeste economische schade door een infectie met M. hyopneumoniae vindt plaats bij de vleesvarkens, maar fokdieren spelen een belangrijke rol in de epidemiologie van de kiem. Ze fungeren namelijk als een permanente bron van M. hyopneumoniae circulatie op het bedrijf en kunnen de nakomelingen reeds op jonge leeftijd besmetten. Om de ziekte en de economische verliezen te controleren, wordt vaccinatie tegen M. hyopneumoniae vaak toegepast op varkensbedrijven. Hierbij worden voornamelijk de fokgelten en de biggen gevaccineerd. Na vaccinatie moet er in het lichaam van het varken een M. hyopneumoniae-specifieke humorale en celgemedieerde immuniteit aanwezig zijn waardoor het dier snel kan reageren wanneer het in contact komt met de kiem. Bovendien wordt de M. hyopneumoniae-specifieke immuniteit overgedragen van zeug naar big via colostrum. Voor de bescherming tegen een M. hyopneumoniae infectie zou de celgemedieerde immuniteit een belangrijkere rol spelen dan de antistoffen in het serum. Echter, de duur van de humorale en celgemedieerde immuniteit opgewerkt door M. hyopneumoniae vaccinatie is onvoldoende onderzocht. Mycoplasma hyopneumoniae-specifieke celgemedieerde immuniteit wordt overgedragen van zeug naar big, maar tot dusver werden de verschillende immuuncellen niet verder gekarakteriseerd.

Diagnostic tools in the field of medicine are required to support examination procedures as performed by practitioners. In Belgium, serological tests still represent the highest number (66%) of all tests performed in pig and cattle industry, followed by bacterial cultures (7%) and molecular assays (3%). The most important reason for this trend is the costs associated with most diagnostic tests as they do not deliver sufficient information within a single test. Even though multiplex quantitative PCRs have been designed to support detection of respiratory disease complexes in cattle and pigs or virotyping of enterotoxigenic Escherichia coli (ETEC), its cost is considered high (approximately 125 EUR) in relation to the delivered information. Over the last decades, various sequencing technologies have been released, allowing a significant reduction in sequencing cost. The availability of real-time single molecule (SMRT) nanopore sequencing (Oxford Nanopore Technologies) initiated a new era in the field of diagnostics (reviewed in Chapter 1). The use of this technology in a metagenomic workflow, allows the co-identification of viral and bacterial pathogens without prior pathogen selection. The added value of this was shown for known respiratory disease complexes in swine and cattle (PRDC and BRDC, respectively), highlighting the co-circulation of primary, secondary, and lesser-known pathogens (e.g., porcine hemagglutinating encephalomyelitis virus (PHEV) and Mycoplasmopsis arginini in PRDC and BRDC, respectively) (Chapter 3). Furthermore, it allowed (semi-)quantitative evaluation of all microbes (both viral and bacterial) to assess their relevance within the disease complex along with sequence information for viruses. The latter is an important asset as it allows viral (sub)typing which is often required to distinguish vaccines from wild type infections (e.g., porcine reproductive and respiratory syndrome virus (PRRSV)) and the subtyping of swine influenza A virus (swIAV). Obtaining these viral genome sequences from a single test also showed to be of great value in surveillance, epidemiology, and tailoring of vaccination as exemplified for porcine parvovirus type 1 (PPV1). Also, in the context of outbreaks it quickly provides sequencing information (e.g., equine herpes virus 1 (EHV-1)). While (m)any host species and sample types (including fecal, serum, respiratory, tissue, and urine) can be subjected to metagenomics, some samples require an additional targeted enrichment to deliver complete viral genomes as shown for swIAV from “dirty” oral fluids (Chapter 4). Costs associated with current tests lie within the range of multiplex PCRs, allowing its use by progressive veterinarians in livestock, companion animals, and exotics, along with various applications for academic institutions and pharmaceutical companies to date. Though, feedback from the field highlighted the need of bacterial virotyping and antimicrobial susceptibility testing (AST) to allow its extended application in routine veterinary diagnostics. This represents a tougher task since existing ad-random metagenomic workflows are not able to deliver sufficient depth on bacterial genomic information yet. Here, a spike-in approach to enrich for specific targets, while maintaining metagenomic sensitivity, is thought to guarantee its wider implementation in routine diagnostic laboratories. Therefore, fundamental knowledge on genetic virulence- and antimicrobial resistance-associated markers should be obtained. To do so, accurate and complete bacterial genomes should be available. Current databases (e.g., NCBI) do not provide enough genomic and phenotypic metadata on veterinary bacteria, thus extensive efforts were made to provide new information for relevant bacterial species. The use of long-read only data for genome assemblies was examined and performed excellent in delivering accurate and complete genomes for non-fastidiously growing bacteria such as E. coli and Actinobacillus species (Chapter 5). This is majorly due to significant improvements in raw read accuracies via new pore chemistries and base calling models over the last years. For fastidiously growing bacterial species, such as Mycoplasmopsis bovis and Brachyspira hyodysenteriae, the generation of a taxon-specific base calling model was shown of great importance to deliver Illumina-level accuracy genomes (>99.999% accuracy) (Chapter 6). Applying genome-wide association studies (GWAS) on these bacterial datasets allowed the identification of various genetic markers (genes, point mutations, and plasmids) with high diagnostic predictive power for virotyping and genetic AST (Chapter 5 and 6). Even though not all resistance phenotypes could be fully explained by these markers, epigenetics (e.g., methylation) along with transcriptomic profiling could still be addressed in the future. For the former, available raw nanopore data could be re-analyzed when more widely applicable methylation software and models are available. This would allow to provide an even broader view on the complex landscape of AMR but will also represent a (very) big challenge to get implemented in a metagenomic context. Prior to the use of these genetic markers in a “virulence and genetic AST enriched” metagenomic workflow, it is recommended to still perform extensive validation via targeted mutagenesis to deliver proof-of-concept for their actual link with virulence and AMR phenotypes (as discussed in Chapter 7). In conclusion, existing metagenomic diagnostic workflows can be enriched to detect genes associated with virulence and AST in a relatively short time frame. First fundaments for its broader application and integration in routine veterinary diagnostic laboratories can be delivered when genetic markers as described in this work will be implemented. A similar approach can be exploited to augment existing metagenomic tools for a wider variety of relevant viruses and bacteria depending on the needs within the field. In the end, this will deliver accurate all-in-one diagnostic tests which will speed-up identification and AST for relevant bacteria in acute infections and support antimicrobial drug tailorship. Diagnostische testen zijn in de diergeneeskunde noodzakelijk om dierenartsen bij het examineren te ondersteunen. In België worden serologische testen nog steeds het vaakst (66%) toegepast in de varkens- en runderindustrie, gevolgd door bacteriële culturen (7%) en moleculaire testen (3%). De belangrijkste reden voor deze trends is de kosten die gepaard gaan met laatstgenoemde testen omdat deze vaak onvoldoende informatie leveren in één test. Ook al werden multiplex en kwantitatieve PCR’s al ontworpen om de detectie van respiratoire ziektecomplexen in runderen en varkens of pathotypering van enterotoxigene Escherichia coli (ETEC) te ondersteunen, de geassocieerde kost is nog steeds te hoog (ongeveer 125 euro per staal). De laatste jaren werden verschillende technologieën voor sequentieanalyse geïntroduceerd, wat toeliet om de kost voor het sequeneren significant te verlagen. Het beschikbaar hebben van de realtime single molecule (SMRT) nanopore technologie (Oxford Nanopore Technologies) opende een nieuw tijdperk in de diagnostiek (gereviewd in Hoofdstuk 1). Het gebruik van deze technologie in een metagenomische workflow laat toe om zowel virale als bacteriële ziekteverwekkers in een enkele test te detecteren zonder vooraf pathogenen te selecteren. De toegevoegde waarde van deze toepassing werd aangetoond voor gekende respiratoire ziektecomplexen bij varken en rund. Dit toonde aan dat er cocirculatie van primaire, secundaire, en minder gekende pathogenen (zoals porcien hemagglutinating encephalomyelitis virus (PHEV) en Mycoplasmopsis arginini respectievelijk bij varken en rund (Hoofdstuk 3)). Daarnaast laat de technologie ook toe om alle microben (zowel viraal als bacterieel) op een (semi-)kwantitatieve manier te evalueren om zo hun relevantie binnen het ziektecomplex te bepalen. Dit samen met sequentieanalyses voor virale genomen. Dat laatste is een belangrijk voordeel omdat dit het (sub)typeren van virussen toelaat, wat vaak noodzakelijk is om een onderscheid te kunnen maken tussen vaccinstammen en wild type infecties (vb. porcien reproductief en respiratoir syndroom virus (PRRSV)) en het subtyperen van influenza A-virus bij varkens (swIAV)). De beschikbaarheid van deze genoomsequenties uit één enkele test laat toe om een bijdrage te leveren bij surveillance, epidemiologie, en het eventueel op punt stellen van vaccinaties zoals voorgesteld voor het porcien parvovirus type 1 (PPV1). Ook in de context van een virale uitbraak kan het gebruikt worden om snel sequentie-informatie te bekomen (vb. equien herpes virus 1 (EHV1)). Zo goed als alle dieren en staaltypes (inclusief fecaal, serum, respiratoir, weefsel en urine) kunnen gebruikt worden voor het sequeneren in een metagenomische context. Sommige staaltypes hebben echter nood aan een extra doelgerichte verrijking om complete virale genomen te bekomen. Dit werd aangetoond voor swIAV uit “vuile” orale vloeistoffen (Hoofdstuk 4). Kosten die geassocieerd worden met huidige testen liggen in de orde van multiplex PCR’s die toelaten gebruikt te worden door progressieve dierenartsen van vee, gezelschapsdieren en exotische dieren, samen met toepassingen voor academische instituten en farmaceutische bedrijven. Feedback uit het veld suggereert dat er nood is aan verdere typering van bacteriën (vb. virotypering) en het bepalen van antimicrobiële gevoeligheid om de huidige test breder bij routinediagnostiek te kunnen toepassen. Dit houdt een grotere opgave in omdat bestaande ad random metagenomische workflows nog niet in staat zijn om voldoende diepgang te leveren voor bacteriële genomische informatie. Hierbij zou een doelgerichte spike-in benadering toelaten om een verrijking uit te voeren voor specifieke genen, met het behoud van metagenomische gevoeligheid. Dit zou een uitgebreidere toepassing bieden voor routinediagnostiek in diagnostische laboratoria. Om dit toe te laten is fundamentele kennis noodzakelijk rond genen die geassocieerd worden met virulentie en antibioticaresistentie, waarvoor accurate en complete bacteriële genomen voorhanden moeten zijn. Huidige databases, zoals NCBI, hebben vaak onvoldoende genomische en fenotypische metadata voor veterinaire bacteriën beschikbaar. Dus werd moeite gedaan om deze informatie te voorzien voor relevante bacteriële species. Het gebruik van lange reads voor genoomassemblage werd uitgebreid getest en kon voor “standaard” bacteriën zoals E. coli en Actinobacillus species excellente genomen met voldoende accuraatheid en compleetheid afleveren (Hoofdstuk 5). Dit is vooral mogelijk door de significante verbetering in accuraatheid van ruwe reads via nieuwe technologie-updates waaronder nanopore chemie en base calling algortimes over de laatste jaren heen. Voor “niet-standaard” bacteriële species, zoals Mycoplasmopsis bovis en Brachyspira hyodysenteriae, was een taxon-specifiek base calling model noodzakelijk om gouden standaardniveau genomen (Illumina; >99.999% accuraatheid) af te leveren (Hoofdstuk 6). Het toepassen van genoom-wijde associatiestudies (GWAS) op deze bacteriële datasets liet toe om verscheidene genetische merkers (genen, puntmutaties en plasmides) te identificeren met hoge diagnostische voorspellende kracht voor zowel virotypering en bepaling van antibioticagevoeligheid (Hoofdstuk 5 en 6). Ook al konden niet alle resistentie fenotypes compleet voorspeld worden, in de toekomst kunnen deze via epigenetica (vb. methylatie) of het profileren van het bacterieel transcriptoom verder onderzocht worden. Voor die eerste kan de beschikbare ruwe nanopore data opnieuw geanalyseerd worden als er meer wijder toepasbare methylatie software en modellen voorhanden zijn. Dit zou toelaten om het complexe landschap van antibioticaresistentie breder te bestuderen. Het zal een (zeer) grote uitdaging zijn om deze data in een metagenomische context en workflow te implementeren. Vooraleer deze genetische merkers in een “virulentie en genetische gevoeligheidsbepaling aangereikte” metagenomische workflow kunnen gebruikt worden, is het aangeraden om nog uitgebreide validaties uit te voeren via doelgerichte mutagenese om zo proof-of-concept te bieden voor de bijdrage van deze merkers tot het virulentie en/of resistentie fenotype (zoals besproken in Hoofdstuk 7). Conclusie: in een relatief korte tijd kunnen metagenomische diagnostische workflows aangereikt worden met genetische markers, die geassocieerd worden met virulentie en antibioticaresistentie. Deze eerste fundamenten kunnen worden getest door de genetische informatie – zoals beschreven in dit werk – te implementeren om zo een breder toepassingsveld en integratie in veterinaire diagnostische laboratoria te bekomen. Een vergelijkbare benadering kan gebruikt worden om bestaande metagenomische workflows op punt te stellen voor een wijdere variëteit aan relevante virussen en bacteriën in het veld. Finaal zal dit ertoe leiden dat bij acute infecties accurate all-in-one diagnostische tests identificatie, typering en genetische antibioticaresistentiebepaling voor relevante bacteriën kunnen versnellen om zo het gebruik van antibiotica beter te sturen.

Honden en katten zijn de populairste huisdieren ter wereld. Beide diersoorten leven al duizenden jaren samen met mensen. Gedurende die tijd heeft de mensheid rassen gecreëerd met behulp van strenge artificiële selectie. Deze selectie heeft vooral bij de hond geleid tot een wijd arsenaal van fysische verschillen. De domesticatie van honden en katten heeft ook voor populatie “bottlenecks” gezorgd, reducties in populatiegroottes. Een gevolg hiervan is een stijging in genetische ziektes en daling in genetische diversiteit. Hoe de genetische gezondheid van onze huisdieren kan worden verbeterd via DNA testen wordt in deze thesis onderzocht. Verschillende manieren waarmee onderzoekers kunnen bijdragen worden besproken in hoofdstuk 1.

Porcine ETEC is an important cause of bacterial diarrheal illness and is regarded as a global health threat for farm animals. Neonatal diarrhea (ND) and post weaning diarrhea (PWD) caused by ETEC result in severe economic losses for the farming industry worldwide due to increased morbidity and mortality and reduced growth rates. Neonatal piglets can be protected by the transfer of maternal antibodies upon vaccination of the sow. However, upon weaning piglets become highly susceptible to ETEC as the passive immunity wanes at weaning. To protect newly weaned piglets against ETEC infections, an oral live bivalent vaccine, Coliprotec® F4/F18 (Elanco GmbH), has been marketed in the EU and other countries since 2017. However, it could not provide complete protection to piglets. Furthermore, the live vaccine has some limitations and associated risks. Therefore, further research into the development of vaccines or alternative strategies is needed. This requires a deeper understanding of the pathogenesis of ETEC and its interaction with its hosts at the molecular level.

Virus discovery is a specific branch of virological research that is focused on the discovery and characterization of new viruses. Since the first descriptions of viruses at the end of the 19th century, the number of known viruses has grown continually, with the speed at which new viruses are being found increasing at an ever-fastening pace. Technological advancements and an improved understanding of virus biology act as catalysts for novel discoveries, which in turn provide new insights that aid in the development of faster, more informative, more reliable and cheaper methodologies for virus research. In the last decade, the development and optimization of high-throughput sequencing technologies has allowed this self-enforcing cycle to culminate in an exponential expansion of the list of known viruses, while simultaneously showing that what is known pales in comparison to what is left to be discovered. Through a series of specific examples, focused on two particular families of RNA viruses that house many important animal pathogens, the families Arteriviridae and Paramyxoviridae, this doctoral thesis aims to provide a contribution to the current knowledge of mammalian virus diversity and to highlight and discuss some of the key advantages and limitations of currently available research tools.

Angiogenesis is defined as the development and rearrangement of blood vessels from already existing vasculature. This process predominantly takes place during embryogenesis, but there are some physiological as well as pathological situations in the adult life where angiogenesis is very active including wound healing and chronic inflammation. Angiogenesis is a highly complex process that is regulated by a variety of stimuli and it is mostly influenced by the cells within the tissue to be vascularized. One of the many cell types involved are macrophages, phagocytosing cells that are mostly known for playing an essential part in the body’s immune system. However, it is becoming increasingly clear that these highly versatile cells play an important function during angiogenesis as well, both in adult and embryonic tissue. Nevertheless, not much is known about their role in the chicken embryo, an established model in the field of angiogenic research. This PhD aimed to investigate the role of chicken embryonic macrophages during the aniogenic process by looking at the impact of macrophage depletion on blood vessel development. This would not only provide more knowledge on the embryo’s endogenous macrophages, but would also open doors to macrophage-related research using the chick embryo chorioallantoic membrane (CAM) angiogenesis assay. Furthermore, as the model is gaining more popularity we wanted to explore possible applications in other fields of research such as tissue engineering. The first chapter of this dissertation is a literature study that provides the necessary scientific background to understand the doctoral research. First, the process of angiogenesis is explained whereby different types of blood vessel formation are also discussed. Then, the macrophage cell type is discussed and how they can play a role in angiogenesis. Lastly, the focus is shifted to the CAM model, a popular model in angiogenesis research. Different aspects of the assay are discussed such as its advantages and disadvantages, some technicalities, and its use in various areas of research including angiogenesis, cancer, and tissue engineering. The second chapter gives an overview of the aims of the PhD. The third chapter describes the development and validation of a technique that allows for the successful depletion of the macrophage population in the chicken embryo. This method could then be used in macrophage-related research in the CAM model. Clodronate liposomes were injected intravenously in the CAM, after which immunohistochemistry and flow cytometry were implemented for a qualitative and quantitative analysis of the macrophage depletion. Staining of the embryonic macrophages was performed with KUL01, a specific chicken macrophage marker. Both techniques revealed that the population of chicken embryonic macrophages was significantly depleted 3 days after intravenous injection of clodronate liposomes in the CAM. Thus, it was confirmed that the intravenous injection of clodronate liposomes is a viable method to deplete the macrophage population in the chick CAM assay. The fourth chapter investigates the effect of the macrophage depletion on the development of blood vessels. To investigate angiogenesis with the CAM assay, collagen type I plugs on a double layer of nylon mesh were placed on top of the CAM. The double layer of mesh was necessary to allow easy and correct quantification of angiogenesis as only the vessels growing above the plane of the top mesh would be counted towards the angiogenic score. Collagen plugs contained either no cells or highly angiogenic human fibrosarcoma (HT1080) cells to ensure that vascularization of the collagen plugs would take place. In both situations, the depletion of macrophages lead to a significant reduction of the vascularization of the collagen plugs. These results indicate that macrophages play a necessary role during angiogenesis. However, by which mechanisms embryonic macrophages are involved in blood vessel development remains to be elucidated. The fifth chapter focuses on the development of experimental protocols that allow the use of the CAM model in vascular permeability research. This would make it possible to investigate the importance of chicken embryonic macrophages in vessel permeability. Vessel leakage was analysed using two methods, the first one being a modified version of the Miles Assay which requires injection of Evan’s Blue dye and analysis of its leakage in embryonic tissue, and the second technique involving injection of fluorescent dextrans and examining their leakage from the CAM vessels over time. However, both techniques failed to detect an effect on vascular leakage even after injection of VEGF, which has been proven to be a potent disruptor of vessel permeability. This indicates that the CAM model may not provide a reliable vessel leakage model, and that more optimization to our approach of researching vascular permeability is necessary. The sixth chapter describes how the CAM assay was employed to explore the vascularization of biomaterials. After all, the vascularization of engineered tissue is necessary for their successful implementation in tissue repair. Although our results show that blood vessels are able to grow in the engineered scaffolds, most of the samples had already lost their scaffold-structure by the time analysis of their vascularization occurred. This was likely due to their biodegradation, which happened quicker than expected. Further optimization of the biomaterials is therefore necessary before additional in vivo experiments can be considered. This preliminary data highlight the advantages of having access to a fast and cheap in vivo model such as the CAM assay. Chapter seven is a general discussion of the entire dissertation whereby the results and described techniques are critically assessed with the help of relevant scientific literature. Our results and experience with the CAM assay show that the model retains its value in angiogenesis and related research. However, it should be noted that there plenty of technical challenges that can greatly influence the outcome of the assay. In conclusion, though, it is safe to say that the CAM assay has great potential as a preclinical model in established and upcoming fields of research. As we have demonstrated that a successful depletion of the macrophage population in the chicken embryo is possible, the model could be implemented in macrophage-related research, not only in relation to angiogenesis but for example also in tumorigenesis. Furthermore, there are definitely opportunities in collaborative research which help ensure that the CAM assay will remain a relevant model for many years to come.

Nowadays, a growing societal and scientific concern exists regarding the widespread occurrence of potential endocrine disrupting compounds (EDCs) in the aquatic environment. The most important potential EDC classes comprise the steroid hormones, plasticizers and plastics additives. Indeed, even at very low concentrations, steroidal EDCs may affect the hormonal system of aquatic organisms, while leaching of plasticizers and plastics additives into the aquatic environment is inevitable as a result of their extensive use in numerous applications. Until now, limited knowledge was available on the presence of steroidal EDCs, plasticizers and plastics additives in the marine environment. Therefore, an urgent need existed to monitor the fate and effects, as well as the environmental and human risks posed by these emerging micropollutants in marine ecosystems. Given our lack of knowledge on the occurrence of EDCs, the main goal of this dissertation was to investigate the prevalence of steroidal EDCs, plasticizers and plastics additives in the marine environment. To this end, new analytical methods targeting a broad range of compounds (n=97) were developed, optimized and validated. Not only instantaneous sampling techniques were examined, but also novel long-term sampling techniques were investigated (i.e.: active and passive sampling). Both were applied extensively in the Belgian Part of the North Sea (BPNS), from 2016 to 2018. Finally, the potential negative effects of the detected concentrations were assessed. Chapter I entails a comprehensive overview on the fate of EDCs in the aquatic environment. Additionally, a summary on the predominant analytical strategies employed for sampling, extraction and detection of EDCs is provided, including a detailed description of the analytical platforms and their capabilities. This chapter ends with the conceptual framework and research objectives of this PhD study, as outlined in research chapters II, III and IV. The aim of chapter II was to develop and validate two accurate ultra-high performance liquid chromatography (UHPLC) coupled to high-resolution mass spectrometry (HRMS) methods for the quantification of steroidal EDCs (n=70), plastics additives and plasticizers (n=27) in seawater. For each target group, the instrumental methods were combined with optimised large-volume solid phase extraction procedures, as it was the goal to detect residues in the marine environment. The developed methods showed excellent performance characteristics (according to 2002/657/EC and Eurachem guidelines) and versatility to fresh water samples. In chapter III, a new sorbent phase for passive sampling purposes was investigated that enables to capture a broad polarity range of emerging organic compounds (log P range between 1.30 to 9.85). For 131 compounds the sampler-water partition coefficients (Ksw) could be determined by using a static exposure design. Calculation of the thermodynamic parameters indicated that the main partitioning process for the (alkyl)phenols, personal care products, pesticides and pharmaceuticals was driven by physisorption, while the uptake of phthalates and steroidal EDCs was mediated by a combination of physisorption and chemisorption. It was clearly demonstrated in this chapter that hydrophilic divinylbenzene can be used for passive sampling. Using the newly developed methodologies from chapters II and III, active and passive samplers were intensively deployed in the Belgian Part of the North Sea, as discussed in chapter IV. The detected concentrations for the steroidal EDCs were mainly below 10 ng L-1, while for the plastics additives and plasticizers concentrations between 10 and 1000 ng L-1 were observed. Concentrations detected by passive sampling, were generally lower than those in active samples. Furthermore, it could be concluded that our proposed strategy for active and passive sampling offers a complementary approach for the measurement of potential EDCs in the marine environment. Ultimately, a risk assessment was performed to investigate the potential risks of the detected compounds to aquatic organisms. Finally, in chapter V, the general discussion and future perspectives of this PhD study are presented. Thereby, it was suggested that future research should focus on the implementation and use of an automized analysis pipeline for suspect and untargeted screening. Also, the development of new passive samplers containing hydrophilic DVB could be promising, as Speedisks showed reduced Ksw as compared to naked sorbent. Moreover, Speedisks could serve as a viable archive tool for sampled EDCs, but this should be investigated more extensively. Finally, a more in-depth evaluation of contaminant levels in Speedisk passive samplers as compared to those in biota could offer an added value towards environmental risk assessment of the findings obtained in this PhD. Overall, this thesis contributes to the establishment of integrated active and passive sampling approaches for monitoring potential EDCs in the marine environment, and more specifically the BPNS.

Zowel bij de hond als het paard is manken door een knieprobleem een frequent voorkomende klacht, maar de specifieke pathologieën, hun oorzaken en hun prevalentie zijn verschillend. De aanwezigheid en uitgebreidheid van deze aandoeningen kan onderzocht worden met verschillende beeldvormingstechnieken zoals radiografie (RX), echografie (US), computer tomografie (CT), computer tomografische arthrografie (CTA) en magnetische resonantie (MRI). Het is essentieel om het onderscheid te kunnen maken tussen normale anatomie, anatomische variaties, toevalsbevindingen en klinisch relevante afwijkingen en dit is nog belangrijker geworden door het toegenomen gebruik en stijgende beschikbaarheid van meer geavanceerde beeldvormingsmodaliteiten. Het hoofddoel van dit doctoraat was om aan de hand van US en CT, de beschrijving bij te werken van de normale anatomie, anatomische variaties en incidentele bevindingen van het kniegewricht bij honden en paarden.