Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

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Consequences of dry period length and dietary energy source on physiological health variables in dairy cows and calves
Mayasari, Nova - \ 2017
Wageningen University. Promotor(en): Bas Kemp, co-promotor(en): Ariette van Knegsel; Henk Parmentier. - Wageningen : Wageningen University - ISBN 9789463431408 - 221
dairy cows - calves - dry period - feed rations - feeds - energy balance - animal health - inflammation - antibodies - adaptation physiology - immunology - melkkoeien - kalveren - gustperiode - voedingsrantsoenen - voer - energiebalans - diergezondheid - ontsteking - antilichamen - adaptatiefysiologie - immunologie

During the transition period, dairy cows experience a negative energy balance (NEB) caused by the high energy requirement for milk yield, while feed intake is limited. Severity of the NEB has been associated with an increased incidence of metabolic disorders and infectious diseases, inflammation, immunosuppression and oxidative stress. It is known that shortening or omitting the dry period or feeding a glucogenic ration improves the energy balance (EB) in dairy cows in early lactation. It can be expected that an improvement of the EB due to shortening or omitting the dry period results in reduced inflammation, immunosuppression and less oxidative stress in dairy cows in early lactation. The first objective of this thesis was to study the effects of dry period length and dietary energy source on immune competence, inflammatory biomarkers and oxidative stress in dairy cows over 2 subsequent lactations. The second objective was to study the consequences of maternal dry period length on colostrum immunoglobulin content and immune competence of calves in the first 12 weeks of life. In the current study, 167 cows were assigned to 3 dry period lengths (0, 30, or 60 d) and 2 early lactation rations (glucogenic or lipogenic). Cows were planned to have the same dry period length and ration over 2 subsequent lactations. Omitting the dry period reduced plasma bilirubin levels compared with a conventional dry period, which is line with the better EB in cows with a 0-d dry period. Effects of dry period length on inflammatory biomarkers, oxidative stress variables and natural antibodies (NAb) titers were, however, less consistent. Omitting the dry period increased not only negative acute phase proteins (APP) in plasma, but also positive APP, oxidative stress variables in plasma, and NAb in milk. Shortening the dry period to 30-d did not influence inflammatory biomarkers and oxidative stress compared with a conventional dry period of 60-d. Occurrence of clinical health problems did not differ between cows with different dry period lengths. In the current study, changes in positive APP and oxidative stress variables in plasma and NAb in milk could be explained by the occurrence of clinical health problems related to inflammation (clinical mastitis, fever, metritis and retained placenta), rather than a better EB due to a shorter or no dry period. Moreover, a higher titer of IgG binding lipopolysaccharide in plasma was associated with decreased odds of high somatic cell count and occurrence of clinical mastitis. In the first lactation after implementation of dry period length and dietary treatments, feeding a glucogenic ration in early lactation increased NAb titers in milk compared with a lipogenic ration, which could be explained partly by a better EB. In the second lactation after implementation of dry period length and dietary treatments, feeding a lipogenic ration in early lactation increased cholesterol levels in plasma compared with a glucogenic ration, which could be related to the high fat content in this ration. Cows with a 0-d dry period had a lower colostrum production and less immunoglobulins in colostrum compared with cows with a 30-d or 60-d dry period. After colostrum uptake, NAb titers in plasma of calves from cows with a 0-d dry period were lower during the first week of life compared with calves from cows with a 30-d or 60-d dry period. Levels of specific antibodies in calves, after immunization in week 6 and 10, in calves were not affected by the maternal dry period length. Birth weight of calves from cows with a 0-d dry period was lower compared with calves from cows with a 30-d dry period, but not compared with calves from cows with a 60-d dry period. Growth of calves until 12 weeks of life was not affected by dry period length. In conclusion, although shortening and omitting the dry period improved the EB in early lactation, this did not result in clear consistent effects of dry period length on inflammation or oxidative stress. Changes in inflammation biomarkers, oxidative stress variables and NAb in milk were a reflection of the occurrence of health problems related to inflammation in particular clinical mastitis and compromised uterine health. Furthermore, albeit omitting the dry period compared with shortening or conventional dry period cows resulted in a reduced immunoglobulin content in colostrum and reduced NAb titers in plasma of their calves in the first week of life, but did not affect specific immune response of the calves in the first 12 weeks of life.

Extensive dry heating-induced changes in physicochemical and immunological properties of whey proteins
Liu, Fahui - \ 2016
Wageningen University. Promotor(en): Tiny van Boekel; Jan Wichers, co-promotor(en): Kasper Hettinga; Gosia Teodorowicz. - Wageningen : Wageningen University - ISBN 9789463430012 - 146
whey protein - heating - physicochemical properties - immune tolerance - antibodies - allergens - wei-eiwit - verwarming - fysicochemische eigenschappen - immunotolerantie - antilichamen - allergenen

Baked milk products, e.g. milk-protein containing muffins or baked cheese, can be tolerated by most cow’s milk allergic subjects. These products were also reported to contribute to the development of immune tolerance in allergic subjects. The main objective of this thesis was to investigate the effects of heating under dry conditions on the physicochemical and immunological properties of whey proteins. A simplified heating model, consisting of whey proteins and lactose, was used to reproduce baking conditions. Most of the Maillard reaction sites were found to be located in the reported conformational epitopes on whey proteins. Therefore, the structural changes subsequently resulted in a decreased IgG-binding capacity. The binding of glycation products to the receptor of AGE (RAGE) increased with heating time. Next, the formation of AGEs was further studied. Formation of sRAGE-binding ligands depended on the aggregation, “pH”, and aw of the samples. Moreover, the sRAGE-binding activity of the samples after digestion was changed and correlated with the digestibility of samples. Based on these results, a correlation between the formation of AGEs and their immunogenicity was hypothesized. Thus, the macrophage immunogenicity of glycated BLG was furtherly studied. The IgE-binding capacity of glycated samples and their influence on the polarization and gene expression of macrophages were studied in vitro. Glycation of BLG was found to reduce the expression of pro-inflammatory TNF-α, and increase the expression of anti-inflammatory TGF-β in M1 and M2 macrophages. The immunomodulatory potential of glycated BLG was further studied, as described. The uptake of glycated BLG by dendritic cells (DCs) was studied. Results showed that glycated BLG inhibited the degranulation of basophils in a dose-dependent manner. Glycation of BLG enhanced its uptake by DCs. However, the degradation of glycated BLG was faster than unheated BLG, indicating a retarded allergen-presentation efficiency of glycated BLG by DCs.

In conclusion, this thesis showed that extensive dry heating induces profound and specific effects on the physicochemical and immunological properties of whey proteins. Conditions during heating, such as aw and “pH”, affect the consequences of heating on whey proteins and their subsequent functions in interacting with immune cells. Compared to unheated and nonglycated samples, glycated BLG can be more efficiently taken up and degraded by DCs. In addition, glycation confers immunomodulatory properties on whey proteins, as tested in macrophages. These results might have consequences for preparing extensively dry heated allergens that can be used in oral immunotherapy. The data in this thesis also provided a better understanding on the mechanism underlying the observation that the development of immune tolerance can be accelerated by baked milk.

Biomarkers and mechanisms of natural disease resistance in dairy cows
Altena, S.E.C. van - \ 2016
Wageningen University. Promotor(en): Huub Savelkoul, co-promotor(en): Edwin Tijhaar. - Wageningen : Wageningen University - ISBN 9789462578005 - 158 p.
dairy cows - biomarkers - disease resistance - immunity - antibodies - proteomics - immune response - dendritic cells - immunology - melkkoeien - ziekteresistentie - immuniteit - antilichamen - eiwitexpressieanalyse - immuniteitsreactie - dendritische cellen - immunologie

The aim of this thesis was to define and test biomarkers for disease resistance in dairy cows and to determine the underlying mechanism in natural disease resistance. The health status of the cows is an important issue in dairy farming. Due to the mandatory reduction in the use of antibiotics, alternatives are required to prevent the development and expression of illness in dairy cows. The identification of biomarkers associated with such disease offers the opportunity to adapt the management of cows at risk, and in this way, prevent them from developing overt disease. Previously, natural antibodies (NAbs) in serum and milk were used as candidate biomarkers for natural disease resistance in cows. In this thesis, we continue on the occurrence and mode of action of NAbs and also focus on their source: the B-1 cells. We performed a literature study on the identification and function of B-1 cells in different species and defined the limitations in the current identification of these cells in pigs, sheep and cows (Chapter 2). B-1 cells were described in cows by using widely accepted cell surface markers CD5 and CD11b. However, in literature several findings suggest that these cell surface markers are not unique markers for B-1 identification. The similarities between mice and veterinary animals in foetal B-cell development and antibody production, implies that B-1 cells are present in cows. In chapter 3, we carefully studied new markers to selectively identify B-1 cells in cows. The combination of B-1 cell markers IgM++ and pSYK++ (indicator constitutive intracellular signalling) identifies a distinct cell population with essential B-1 characteristics such as high CD80 expression. In addition, the development of these B-1 cells in calves before colostrum intake and 3 weeks afterwards shows the same kinetics as the development of NAbs represented by IgM antibodies binding to the well-accepted NAb-antigen phosphatidylcholine (PtC). In calves up to half a year of age, it is shown that the production of such NAbs increases from birth and stabilises from 6 weeks onwards. This implies an endogenous NAb production, which follows the same age-related kinetics as can be expected from B-1 cell development. In contrast, the development of total IgM antibody levels in calves shows a bimodal distribution, which is caused by the uptake and breakdown of maternally-derived IgM and simultaneous endogenous production of specific and natural IgM. Chapter 4 describes the role of such NAbs in bovine immunity. NAbs were represented by the binding of IgM to the naïve antigen keyhole limpet hemocyanin (KLH). Cows with high serum NAb levels were shown to have more IgM and IgG antibodies binding to common microbial structures LPS, LTA and PGN, than cows with low serum NAb levels. In addition, they also have more IgM antibodies binding to intact, fixed E. coli and S. Typhimurium bacteria. However, the killing of live E. coli and S. Typhimurium bacteria via antibody-mediated complement killing does not differ between cows with high and low NAb levels. The antibody-mediated complement killing was determined in a newly developed serum bactericidal test. Cows that performed less in the bactericidal test were more likely to develop mastitis in the future. This association was observed for the killing of E. coli and S. Typhimurium and the development of mastitis within the next one year. For S. Typhimurium the association was still present for the cases of mastitis occurring within four years after testing. Alternative biomarkers for disease resistance in cows were defined in chapter 5 by using a contemporary proteomics approach. Milk samples from high and low disease resistant cows were selected from the “Resilient Cattle” (Weerbaar Vee) biobank. Comparing the spectrum of milk proteins of high and low disease-resistant cows showed potential candidate biomarkers that were elevated in the milk of low-resistant cows. Two candidate marker proteins were validated with ELISA in a new and larger group of high- and low-resistant cows. Lactoferrin (LF) levels were significantly increased in milk of low-resistant cows. In addition, LF levels in milk were associated with clinical manifestations of lameness and had a predictive value for subsequent culling.

In conclusion, we found that also in cows NAbs are produced by B-1 cells that can be identified based on the combined expression of cell surface IgM and internal pSYK. In addition, the frequency of these B-1 cells after birth follows a similar kinetics as described before in mice. These NAbs can be more precisely identified based on their PtC binding ability and their functional activity in a bactericidal test. However, the true predictive value of B-1 cells and NAbs for the health status and immunocompetence of dairy cattle remains to be established. Proteomics turned out to be a useful approach for identifying potential new biomarkers for health and disease in milk of cows. Application and further development of their predictive capacity is dependent on the availability of robust, sensitive and quantitative assays. This project was part of the “Resilient Cattle” project providing biological samples and essential data on the health status during respective lactation periods of individual dairy cows. The impact of this research now requires translation into management tools and principles for the individual farmer impacting on the overall health status and economic performance of his herd of dairy cattle.

Capture of Tumor Cells on Anti-EpCAM-Functionalized Poly(acrylic acid)-Coated Surfaces
Andree, Kiki C. ; Barradas, Ana M.C. ; Nguyen, Ai T. ; Mentink, Anouk ; Stojanovic, Ivan ; Baggerman, Jacob ; Dalum, Joost van; Rijn, Cees J.M. van; Terstappen, Leon W.M.M. - \ 2016
ACS Applied Materials and Interfaces 8 (2016)23. - ISSN 1944-8244 - p. 14349 - 14356.
antibodies - circulating tumor cells - epithelial cell adhesion molecule (EpCAM) - flow model - polycarboxylate coating - surface plasmon resonance

The presence of tumor cells in blood is predictive of short survival in several cancers and their isolation and characterization can guide toward the use of more effective treatments. These circulating tumor cells (CTC) are, however, extremely rare and require a technology that is sufficiently sensitive and specific to identify CTC against a background of billions of blood cells. Immuno-capture of cells expressing the epithelial cell adhesion molecule (EpCAM) are frequently used to enrich CTC from blood. The choice of bio conjugation strategy and antibody clone is crucial for adequate cell capture but is poorly understood. In this study, we determined the binding affinity constants and epitope binding of the EpCAM antibodies VU1D-9, HO-3, EpAb3-5, and MJ-37 by surface plasmon resonance imaging (SPRi). Glass surfaces were coated using a poly(acrylic acid) based coating and functionalized with anti-EpCAM antibodies. Binding of cells from the breast carcinoma cell line (SKBR-3) to the functionalized surfaces were compared. Although EpAb3-5 displayed the highest binding affinity HO-3 captured the highest amount of cells. Hence we report differences in the performance of the different antibodies and more importantly that the choice of antibody to capture CTC should be based on multiple assays.

Antibodies and longevity of dairy cattle : genetic analysis
Klerk, B. de - \ 2016
Wageningen University. Promotor(en): Johan van Arendonk, co-promotor(en): Jan van der Poel; Bart Ducro. - Wageningen : Wageningen University - ISBN 9789462577589 - 134 p.
dairy cattle - dairy cows - antibodies - longevity - genetic analysis - breeding value - genomes - genetic improvement - animal genetics - melkvee - melkkoeien - antilichamen - gebruiksduur - genetische analyse - fokwaarde - genomen - genetische verbetering - diergenetica

The dairy sector has a big impact on food production for the growing world population and contributes substantially to the world economy. In order to produce food in a sustainable way, dairy cows need to be able to produce milk without problems and as long as possible. Therefore, breeding programs focuses on improvement of important traits for dairy cows. In order to improve desirable traits and obtain genetic gain there is a constant need for optimization of breeding programs and search for useful parameters to include within breeding programs. Over the last several decades, breeding in dairy cattle mainly focused on production and fertility traits, with less emphasis on health traits. Health problems, however, can cause substantial economic losses to the dairy industry. The economic losses, together with the rising awareness of animal welfare, increased herd size, and less attention for individual animals, have led to an increased need to focus more on health traits. Longevity is strongly related to disease resistance, since a more healthy cow will live a longer productive life (longevity). The identification of biomarkers and the detection of genes controlling health and longevity, would not only greatly enhance the understanding of such traits but also offer the opportunity to improve breeding schemes. The objectives of this thesis therefore were 1) to find an easy measurable disease resistance related biomarker in dairy cows, 2) identify the relation between antibodies and longevity, 3) identify genomic regions that are involved with antibody production/expression. In this thesis antibodies are investigated as parameter for longevity. Antibodies might be a novel parameter that enables selection of cows with an improved ability to stay healthy and to remain productive over a longer period of time. In this thesis antibodies bindiging the naive antigen keyhole limpet hemocyanin (KLH) were assumed to be natural antibodies. Antibodies binding bacteria-derived antigens lipoteichoic acid (LTA), lipopolysaccharide (LPS) and peptidoglycan (PGN) were assumed to be specific antibodies. In chapter 2 it was shown that levels of antibodies are heritable (up to h2 = 0.23). Additionally, antibody levels measured in milk and blood are genetically highly correlated (± 0.80) for the two studied isotypes (IgG and IgM). On the other hand, phenotypically, natural antibodies (from both IgG and IgM isotype) measured in milk cannot be interpreted as the same trait (phenotypic correlation = ± 0.40). In chapter 3 and 4 it was shown that levels of antibodies (both natural-and specific antibodies) showed a negative relation with longevity: first lactation cows with low IgM or IgG levels were found to have a longer productive life. When using estimated breeding values for longevity, only a significant relation was found between natural antibody level (IgM binding KLH) and longevity. Lastly chapter 5 reports on a genome-wide-association study (GWAS), to detect genes contributing to genetic variation in natural antibody level. For natural antibody isotype IgG, genomic regions with a significant association were found on chromosome 21 (BTA). These regions included genes have impact on in isotype class switching (from IgM to IgG). The gained knowledge on relations between antibodies and longevity and the gained insight on genes responsible for natural antibodies level make antibodies potential interesting biomarkers for longevity.

Biofunctionalized nanoporous aluminum oxide culture chips : for capture and growth of bacteria
Debrassi, A. - \ 2016
Wageningen University. Promotor(en): Han Zuilhof; Willem de Vos; Tom Wennekes. - Wageningen : Wageningen University - ISBN 9789462576179 - 218 p.
aluminium oxide - porous media - unimolecular films - immobilization - bacteria - binding - antibodies - aluminiumoxide - poreus medium - unimoleculaire films - immobilisatie - bacteriën - binden - antilichamen

Porous aluminum oxide (PAO) is a nanostructured material used for various biotechnological applications, including the culturing microorganisms and other types of cells. The ability to chemically modify the PAO surface and tailor its surface properties is a promising way to expand and refine its applications. The immobilization of biomolecules on PAO that specifically interact with and bind to target bacteria would enable the capture and subsequent growth of bacteria on the same surface, and this was the ultimate goal of the research presented in this thesis.

After a general introduction to the overall subject of this thesis, presented in Chapter 1, the most commonly used and recent methods to prepare glycosurfaces are reviewed and compared on their merits and drawbacks in Chapter 2. Although there are a great number of techniques, the main challenge that still remains is to develop an accessible, reproducible and inexpensive approach that produces well-defined and stable glycosurfaces using as few steps as possible. The most used analytical techniques for the characterization of glycosurfaces and several applications of these surfaces in the binding, capture, and sensing of bacteria and bacterial toxins were also discussed in Chapter 2.

Biofunctionalization of surfaces in general requires a stepwise approach, in which it is very important to have a stable monolayer as the first step. At the beginning of this research it was known that various functional groups were able to react with (porous) aluminum oxide, but there was no comprehensive study comparing the stability of these modified surfaces under the conditions that are important for microbiological applications. In Chapter 3, the PAO surface was modified with various functional groups known to react with PAO (carboxylic acid, α-hydroxycarboxylic acid, alkyne, alkene, phosphonic acid, and silane), and the stability of these modified surfaces was assessed over a range of pH and temperatures that are relevant for microbial growth. Silane and phosphonate-modified PAO surfaces with a hydrophobic monolayer proved to be the most stable ones, but the phosphonate modification was both more easily applied and reproducible. This modification was stable for at least two weeks in buffer solutions with pH values between 4 and 8, and at temperatures up to 40 °C. Only at elevated temperatures of 60 °C and 80 °C under hydrolytic conditions it was observed that the stability of the same monolayer on PAO decreased gradually. As a proof-of-principle for the biofunctionalization and bacterial capture on this PAO phosphonate monolayer, an alkyne-terminated monolayer was biofunctionalized via a CuAAC click reaction with an azido-mannoside and the binding and growth of Lactobacillus plantarum was successfully demonstrated.

In Chapter 4 various approaches to install reactive groups onto the phosphonate-modified PAO surface were developed, creating a (bio)functionalization “tool-box”. PAO surfaces presenting different terminal reactive groups were prepared, such as azide, alkyne, alkene, thiol, isothiocyanate, and N-hydroxysuccinimide (NHS), starting from a single, straightforward and stable initial modification with a bromo-terminated phosphonic acid. These reactive surfaces were then used to immobilize (bio)molecules, including carbohydrates and proteins. Fluorescently labeled bovine serum albumin (BSA) was covalently immobilized on the PAO surface as a proof-of-principle, and it was shown that a range of bacteria could still grow on the BSA-functionalized PAO surface.

With a PAO (bio)functionalization tool-box in hand, the successful proof-of-principle mannoside-dependent binding and growth of L. plantarum on PAO (Chapter 3) was further investigated and expanded upon (Chapter 5). The parameters involved in the preparation of these surfaces and in the binding with L. plantarum were investigated in more detail in Chapter 5, such as the nature of the spacer connected to the mannoside derivative and the presence of soluble carbohydrates and bovine serum albumin (BSA) in the medium. The surfaces with the azido-mannoside with the long hydrophobic spacer showed the best binding of L. plantarum when compared to a long PEG-based hydrophilic spacer and a short hydrophobic one. The presence of a soluble a-glucoside did not prevent the binding of the bacteria to the mannose-presenting PAO, and similar results were obtained when BSA was present. Additionally, a mutant strain of L. plantarum that does not have the mannose-specific adhesion was not able to bind to the mannose-presenting PAO. When taken together, this proves that the mannoside–adhesin interaction is the main mechanism of binding the bacteria to the mannose-biofunctionalized PAO in this system.

In Chapter 6, the NHS-terminated PAO developed in Chapter 4 was used for the immobilization of antibodies against Escherichia coli. After an extensive optimization of the modification chemistry of the surfaces and the incubation conditions, commercially available anti-E. coli antibodies were immobilized on the PAO surface. Binding and washing experiments indeed demonstrated increased binding of E. coli on the antibody-presenting PAO surfaces, providing avenues for testing other bacteria such as Lactobacillus rhamnosus GG widely used in probiotic formulations worldwide.

In Chapter 7, the most important achievements of this project are discussed, together with additional ideas and recommendations for further research. Most notably some preliminary results are presented on the immobilization of two antibodies against L. rhamnosus GG: anti-L. rhamnosus GG, against the whole bacterial cell, and anti-SpaC, against only the SpaC part of the pili present on the cell surface of L. rhamnosus GG. Anti-L. rhamnosus GG antibody showed promising but not yet optimal increased binding of L. rhamnosus GG. Finally, some reflections on PAO and its (bio)functionalization are provided in the context of a risk analysis and technology assessment.

Effect of dry period length and dietary energy source in dairy cows on natural antibody titers and somatic cell count in milk.
Mayasari, N. ; Rijks, W. ; Vries Reilingh, G. de; Remmelink, G.J. ; Kemp, B. ; Parmentier, H.K. ; Knegsel, A.T.M. van - \ 2015
dry period - somatic cell count - antibodies
Omission of the dry period of cows improved energy balance (EB) and showed variable effects on somatic cell counts (SCC) and natural antibodies (NAb) in milk. A glucogenic diet compared with lipogenic diet enhanced plasma NAb binding keyhole limpet hemocyanin (KLH). NAb in milk were associated with SCC. It was hypothesized that during negative energy balance, NAb either have a role or reflect in inflammatory processes and are associated with SCC. The objective was to study effects of dry period length and dietary energy source on titers of NAb binding KLH and lipopolysaccharide (LPS) in milk, SCC and mastitis. In total, 167 Holstein-Friesian dairy cows were randomly assigned to treatments. Treatments consisted of 3 dry period lengths: 0-, 30- or 60-d and 2 early lactation diets (glucogenic or lipogenic), in a 3 × 2 factorial design. Cows enrolled in this study were clinically healthy and had SCC in milk < 250,000 cell/mL. Milk samples for NAb and SCC measurement were sampled weekly and 4 times per week, respectively, from wk 1 until 14 postpartum. The data collected were statistically analyzed using ANOVA and logistic regression. Cows with a 0-d dry period had higher titers of IgG and IgM binding KLH and LPS and higher SCC in milk compared with cows with a 30- or 60-d dry period (P < 0.01). Mastitis incidence was 17% and did not differ between dry period lengths or lactation diets. A glucogenic diet showed higher titers of IgM binding LPS and tended to have higher titers of IgG binding LPS in milk compared with a lipogenic diet (P < 0.01 and P = 0.08, respectively). Higher titers of IgG and IgM binding KLH and IgG binding LPS were associated with increased risk of high SCC (P < 0.05). Higher IgG and IgM binding KLH and LPS were also associated with increased risk of mastitis (P < 0.05). The results demonstrate that cows with a 0-d dry period and fed a glucogenic diet showed high titers of IgG binding LPS in milk. Moreover, we can conclude that IgG and IgM binding KLH or LPS in milk might be additional valuable tools to detect increased risks for mastitis in dairy cows.
Linking early life conditions to osteochondrosis prevalence in gilts
Koning, D.B. de - \ 2015
Wageningen University. Promotor(en): Bas Kemp, co-promotor(en): Ilse van Grevenhof; Wouter Hazeleger. - Wageningen : Wageningen University - ISBN 9789462575042 - 234
gelten - osteochondritis - varkensvoeding - vloertypen - bouw (dier) - voortbeweging - antilichamen - koolhydraatrijk voedsel - arginine - voedersupplementen - diergezondheid - gilts - pig feeding - floor type - conformation - locomotion - antibodies - carbohydrate-rich foods - feed supplements - animal health

Osteochondrosis (OC) involves the development of necrotic growth cartilage near the surface of a joint and is suggested to be associated with lameness in sows. Development of OC in pigs occurs at young age in a relatively short time frame of several weeks around 10 weeks of age. Due to this time dependency, one may wonder whether there are time dependent effects of factors or conditions associated with OC. The aim of this thesis was to assess whether OC prevalence is associated with conditions encountered in early life such as dietary restriction, floor type, conformation and locomotive characteristics (CLC), natural (auto-) antibodies (N[A]Ab), and carbohydrate levels. Indications for time dependent effects were found for dietary restriction on OC prevalence. This indicated that gilts receiving restricted feeding from 4 to 10 weeks of age and switched to ad libitum feeding until 26 weeks of age had a significantly higher prevalence of OC when compared to gilts receiving restricted feeding after 10 weeks of age. Time dependent effects of floor type were not clearly present, but gilts housed on a deep litter type system using wood shavings after weaning had a higher prevalence of severe OC when compared to gilts kept on a concrete partially slatted floor. Feed with a lower carbohydrate level increased OC prevalence compared to feed with a higher carbohydrate level. We hypothesized that the effects of dietary restriction, floor type, and dietary carbohydrate levels were mediated through loading of the joints either by, respectively, a short rapid increase in weight gain, higher incidence of play behaviors, or by an overall increased body weight. Although OC has been suggested to be associated with various CLC such as lameness, a consistent association of CLC at young age with OC at slaughter could not be found. This is likely due to CLC and OC both varying over time, making associations between the 2 entities complicated. There were indications that a component of the immune system is associated with OC as N(A)Ab against several antigens were found after weaning in association with OC. However, as with the CLC, associations with OC were not consistent over time, making it difficult to discern the exact associations between N(A)Ab and OC. To conclude, several early life conditions were found to affect or be associated with OC prevalence in gilts. This indicates that if one wants to reduce OC prevalence, one needs to start early after weaning. However, the exact implications of OC on CLC such as lameness and, therefore, welfare remain uncertain and require further studies into the long term welfare effects of OC.

New applications of the interaction between diols and boronic acids
Duval, F.L. - \ 2015
Wageningen University. Promotor(en): Han Zuilhof, co-promotor(en): Teris van Beek. - Wageningen : Wageningen University - ISBN 9789462574717 - 131
antilichamen - immobilisatie - boorzuur - biomarkers - vloeistofchromatografie - antibodies - immobilization - boric acid - liquid chromatography

Florine Duval - New applications of the interaction between diols and boronic acids – Summary

Chapter 1 introduces the theory and known applications of the interaction between boronic acids and diols, and explains the context of this thesis. Diagnosis of depression was the initial goal of this multidisciplinary project. The focus of the PhD project was the development of a strategy to immobilize antibodies on the surface of a chip in such a way that very low concentrations (~ 1 pM) of biomarkers for depression could be detected in urine. To achieve this, the immobilization of antibodies using boronic acids seemed promising.

However, preliminary experiments and further insights revealed the many challenges that this immobilization strategy faces, giving rise to Chapter 2. This chapter discusses several important points that need to be taken into account when one plans to immobilize antibodies via boronic acids: choice of the boronic acid structure and spacer to attach it to the surface, use of an antifouling polymer, choice of an antibody with suitable glycosylation, optimization of the conditions for antibody immobilization...

One big issue for antibody immobilization using boronic acids is the reversibility of the reaction between boronic acids and diols, hence the possible release of the antibody from the surface.

Chapter 3 describes the design and synthesis of boronic acid-containing linkers that would enable the oriented and irreversible immobilization of antibodies. Two linkers were designed with an amine for surface attachment, a boronic acid for capturing antibodies via the N-glycans in their Fc chain, and a diazirine for irreversible immobilization upon UV irradiation while maintaining antibody orientation. From a diazirine building-block that was obtained in three steps, the first linker was synthesized in four steps and the second linker was synthesized in three steps. Diol-functionalized silica was used for the chromatography of two boronic acid-containing intermediates, this method being novel (to the best of our knowledge) and likely based on boronic acid-diol interactions. High-resolution mass spectrometry, through matching exact masses, matching isotope patterns and observation of species corresponding to the esterification of boronic acids with MeOH, confirmed that both linkers were synthesized successfully.

During the synthesis of boronic acid-containing linkers, it was difficult to see which spots on TLC plates corresponded to boronic acids. To solve this problem, a new TLC staining method based on the reaction between boronic acids and alizarin was developed.

Chapter 4 presents this work in detail. After optimization experiments, 1 mM alizarin in acetone was shown to be the preferred staining solution. When the TLC plate was briefly dipped in this solution, allowed to dry in ambient air and observed under 365 nm light, bright yellow fluorescent spots were observed where boronic acids were present. Phenylboronic acid was detected at a concentration as low as 0.1 mM. A range of boronic acids and derivatives was successfully detected, and boron-free compounds resulted in no or very weak fluorescence. The staining method was further tested in the monitoring of three reactions involving boronic acids, and provided clear information about the consumption or formation of boronic acid-containing compounds.

Although TLC is useful to synthetic chemists, analysis of reaction mixtures by HPLC is sometimes necessary for obtaining more accurate information or for optimization of preparative HPLC conditions.

Chapter 5 presents the development and applicability of a method for the on-line HPLC detection of boronic acids using alizarin. After optimization experiments at an HPLC flow rate of 0.40 mL/min, the HPLC-separated analytes were mixed post-column with a solution of 75 μM alizarin and 0.1% triethylamine in ACN, which was delivered at a flow rate of 0.60 mL/min. The reaction between alizarin and boronic acids occurred in a reaction coil of dimensions of 3.5 m × 0.25 mm at a temperature of 50 °C, resulting in fluorescent complexes that were detected as positive peaks by a fluorescence detector (lexc 469 nm and lem 610 nm). The method enabled the selective detection of various boronic acids and derivatives, with a limit of detection of phenylboronic acid of 1.2 ng or 1 μM. It could successfully monitor the progress of two organic reactions involving boronic acid-containing compounds, and provided useful insights into the course of the reactions.

Chapter 6 provides a reflexion about the work presented in this thesis, suggestions for future research, and a general conclusion.

Allergen-specific cytokine polarization protects shetland ponies against culicoides obsoletus-induced insect bite hypersensitivity
Meulenbroeks, C. ; Lugt, J.J. van der; Meide, N.M.A. van der; Willemse, T. ; Rutten, V.P.M.G. ; Zaiss, D.M.W. - \ 2015
PLoS ONE 10 (2015)4. - ISSN 1932-6203 - 12 p.
e-bearing cells - mast-cells - british-columbia - friesian horses - skin biopsies - sweet itch - ige - antibodies - tolerance - immunotherapy
The immunological mechanisms explaining development of an allergy in some individuals and not in others remain incompletely understood. Insect bite hypersensitivity (IBH) is a common, seasonal, IgE-mediated, pruritic skin disorder that affects considerable proportions of horses of different breeds, which is caused by bites of the insect Culicoides obsoletus (C. obsoletus). We investigated the allergen-specific immune status of individual horses that had either been diagnosed to be healthy or to suffer of IBH. Following intradermal allergen injection, skin biopsies were taken of IBH-affected and healthy ponies and cytokine expression was determined by RT-PCR. In addition, allergen-specific antibody titers were measured and cytokine expression of in vitro stimulated, allergen-specific CD4 T-cells was determined. 24 hrs after allergen injection, a significant increase in mRNA expression of the type-2 cytokine IL-4 was observed in the skin of IBH-affected Shetland ponies. In the skin of healthy ponies, however, an increase in IFN¿ mRNA expression was found. Analysis of allergen-specific antibody titers revealed that all animals produced allergen-specific antibodies, and allergen-specific stimulation of CD4 T-cells revealed a significant higher percentage of IFN¿-expressing CD4 T-cells in healthy ponies compared to IBH-affected ponies. These data indicate that horses not affected by IBH, in contrast to the so far established dogma, are not immunologically ignorant but have a Th1-skewed allergen-specific immune response that appears to protect against IBH-associated symptoms. To our knowledge this is the first demonstration of a natural situation, in which an allergen-specific immune skewing is protective in an allergic disorder.
A cohort study on Actinobacillus pleuropneumoniae colonisation in suckling piglets
Tobias, T.J. ; Klinkenberg, D. ; Bouma, A. ; Broek, J. van den; Daemen, A.J.J.M. ; Wagenaar, J.A. ; Stegeman, J.A. - \ 2014
Preventive Veterinary Medicine 114 (2014)3-4. - ISSN 0167-5877 - p. 223 - 230.
infection patterns - pigs - antibodies - transmission - efficacy - herds - sows - serotype-2 - hemolysin - vaccine
Actinobacillus pleuropneumoniae causes respiratory disease in pigs and despite the use of preventive measures such as vaccination and antimicrobials clinical outbreaks still occur. At weaning often many piglets are not colonised. If differences in prevalence between litters are large and if factors were known that could explain these differences, this may provide an opportunity to raise groups of A. pleuropneumoniae free piglets. To this end, a cohort study was performed on two endemically infected farrow-to-finish farms. Seventy-six of 133 sows were selected using stratified random selection by parity. Farmers complied with a strict hygiene and animal management protocol to prevent transmission between litters. Tonsil brush and serum samples taken three weeks before parturition were tested for antigen with an apxIVA qPCR and antibodies with Apx and Omp ELISAs, respectively. Three days before weaning tonsil brush samples from all piglets (n = 871) were collected and tested for antigen. Whereas all sows tested positive both in serology tests as well as qPCR, 0.41 of the litters tested fully negative and 0.73 of all piglets tested negative. The proportion of positively tested piglets in positive litters ranged from 0.08-1.0 (median= 0.36). A grouped logistic regression model with a beta binomial distribution of the probability for piglets to become infected was fitted to the data and associations with explanatory variables were explored. To test the possibility that alternatively the clustering was caused by onwards transmission among the piglets, a transmission model was fitted to the data incorporating sow-piglet and piglet-piglet transmission, but this model did not fit better. The results of this study showed that the number of colonised suckling piglets was highly clustered and mainly attributable to the variability of infectiousness of the dam, but no dam related risk factor for colonisation status of litter or piglets within litters could be identified. (C) 2014 Elsevier B.V. All rights reserved.
Getting more out of less - A quantitative Serological Screening Tool for simultaneous detection of multiple influenza A Hemagglutinin-types in chickens.
Freidl, G.S. ; Bruijn, E. de; Beek, J. van; Reimerink, J. ; Wit, S.J. de; Koch, G. ; Vervelde, L. ; Ham, H.J. van der; Koopmans, M.P.G. - \ 2014
PLoS ONE 9 (2014)9. - ISSN 1932-6203
virus-infection - microsphere immunoassay - cross-protection - t-lymphocytes - antibodies - subtypes - responses - epitope - h5
Current avian influenza surveillance in poultry primarily targets subtypes of interest for the veterinary sector (H5, H7). However, as virological and serological evidence suggest, surveillance of additional subtypes is important for public health as well as for the poultry industry. Therefore, we developed a protein microarray enabling simultaneous identification of antibodies directed against different HA-types of influenza A viruses in chickens. The assay successfully discriminated negative from experimentally and naturally infected, seropositive chickens. Sensitivity and specificity depended on the cut-off level used but ranged from 84.4% to 100% and 100%, respectively, for a cut off level of =1:40, showing minimal cross reactivity. As this testing platform is also validated for the use in humans, it constitutes a surveillance tool that can be applied in human-animal interface studies.
Bluetongue virus without NS3/NS3a expression is not virulent and protects against virulent bluetongue virus challenge.
Feenstra, F. ; Gennip, H.G.P. van; Maris-Veldhuis, M.A. ; Verheij, E. ; Rijn, P.A. van - \ 2014
Journal of General Virology 95 (2014)Pt. 9. - ISSN 0022-1317 - p. 2019 - 2029.
vaccinated animals - rna segment - serotype 8 - sheep - antibodies - particles - europe - cattle - ns3 - differentiation
Bluetongue is a disease in ruminants caused by the bluetongue virus (BTV), and is spread by Culicoides biting midges. Bluetongue outbreaks cause huge economic losses and death in sheep in several parts of the world. The most effective measure to control BTV is vaccination. However, both commercially available vaccines and recently developed vaccine candidates have several shortcomings. Therefore, we generated and tested next-generation vaccines for bluetongue based on the backbone of a laboratory-adapted strain of BTV-1, avirulent BTV-6 or virulent BTV-8. All vaccine candidates were serotyped with VP2 of BTV-8 and did not express NS3/NS3a non-structural proteins, due to induced deletions in the NS3/NS3a ORF. Sheep were vaccinated once with one of these vaccine candidates and were challenged with virulent BTV-8 3 weeks after vaccination. The NS3/NS3a knockout mutation caused complete avirulence for all three BTV backbones, including for virulent BTV-8, indicating that safety is associated with the NS3/NS3a knockout phenotype. Viraemia of vaccine virus was not detected using sensitive PCR diagnostics. Apparently, the vaccine viruses replicated only locally, which will minimize spread by the insect vector. In particular, the vaccine based on the BTV-6 backbone protected against disease and prevented viraemia of challenge virus, showing the efficacy of this vaccine candidate. The lack of NS3/NS3a expression potentially enables the differentiation of infected from vaccinated animals, which is important for monitoring virus spread in vaccinated livestock. The disabled infectious single-animal vaccine for bluetongue presented here is very promising and will be the subject of future studies.
Evaluation of a diagnostic ELISA for insect bite hypersensitivity in horses using recombinant Obsoletus complex allergens
Meide, N.M.A. van der; Savelkoul, H.F.J. ; Meulenbroeks, C. ; Ducro, B.J. ; Tijhaar, E.J. - \ 2014
The Veterinary Journal 200 (2014)1. - ISSN 1090-0233 - p. 31 - 37.
genome-wide association - culicoides-obsoletus - plasma-cells - equine ige - antibodies - responses - expression - extract - skin - ceratopogonidae
Culicoides spp. of the Obsoletus complex belong to the most important species of midge, involved in causing insect bite hypersensitivity (IBH) in horses in The Netherlands. The aim of the current study was to evaluate seven different Obsoletus complex-derived recombinant allergens (Cul o 1–Cul o 7) and to compare these with Obsoletus complex whole body extract (WBE) in an IgE ELISA, using sera of 194 clinically-confirmed cases of IBH and 175 unaffected horses. The highest test accuracy was obtained with WBE, followed by Cul o 2, 3 and 5. Two ELISAs with a combination of recombinant allergens, Combi-1 (Cul o 3, 5 and 7) and Combi-2 (Cul o 1, 2, 5 and 7) were additionally performed and both resulted in high test accuracies close to that obtained with WBE. Combi-1 resulted in the best sensitivity and specificity, both 89%. Both Combi-1 and Combi-2 performed less well with samples collected in winter, but over 70% of the IBH-affected horses could still be identified. In conclusion, a combination of three Obsoletus complex recombinant allergens (Cul o 3, 5 and 7) could potentially replace Obsoletus complex WBE in an IgE ELISA for diagnosis of IBH in horses.
Expression of natural human b1,4-GalT1 variants and of non-mammalian homologues in plants leads to differences in galactosylation of N-glycans
Hesselink, T. ; Rouwendal, G.J.A. ; Henquet, M.G.L. ; Florack, D.E.A. ; Helsper, J.P.F.G. ; Bosch, H.J. - \ 2014
Transgenic Research 23 (2014)5. - ISSN 0962-8819 - p. 717 - 728.
golgi-apparatus - murine beta-1,4-galactosyltransferase - beta 1,4-galactosyltransferase - transgenic plants - gene - cells - localization - antibodies - oligosaccharides - glycoproteins
b1,4-Galactosylation of plant N-glycans is a prerequisite for commercial production of certain biopharmaceuticals in plants. Two different types of galactosylated N-glycans have initially been reported in plants as the result of expression of human b1,4-galactosyltransferase 1 (GalT). Here we show that these differences are associated with differences at its N-terminus: the natural short variant of human GalT results in hybrid type N-glycans, whereas the long form generates bi-antennary complex type N-glycans. Furthermore, expression of non-mammalian, chicken and zebrafish GalT homologues with N-termini resembling the short human GalT N-terminus also induce hybrid type N-glycans. Providing both non-mammalian GalTs with a 13 amino acid N-terminal extension that distinguishes the two naturally occurring forms of human GalT, acted to increase the levels of biantennary galactosylated N-glycans when expressed in tobacco leaves. Replacement of the cytosolic tail and transmembrane domain of chicken and zebrafish GalTs with the corresponding region of rat a2,6-sialyltransferase yielded a gene whose expression enhanced the level of bi-antennary galactosylation even further.
GM1-derived carbohydrates for pathogen and antibody detection : synthesis and biological evaluation
Garcia Hartjes, J. - \ 2014
Wageningen University. Promotor(en): Han Zuilhof, co-promotor(en): Tom Wennekes. - Wageningen : Wageningen University - ISBN 9789462571273 - 240
koolhydraten - moleculaire detectie - antilichamen - pathogenen - remmers - biotesten - carbohydrates - molecular detection - antibodies - pathogens - inhibitors - bioassays
Factors associated with Culicoides Obsoletus complex spp.-specific IgE reactivity in Icelandic horses and Shetland ponies
Schurink, A. ; Meide, N.M.A. van der; Savelkoul, H.F.J. ; Ducro, B.J. ; Tijhaar, E.J. - \ 2014
The Veterinary Journal 201 (2014). - ISSN 1090-0233 - p. 395 - 400.
insect bite hypersensitivity - equine ige - netherlands - antibodies - diagnosis - allergens - extract
Insect bite hypersensitivity (IBH) is a common allergic skin disease in horses, caused by biting insects of the Culicoides spp. In The Netherlands, Culicoides spp. of the Obsoletus complex are the most important midges involved in IBH. The aim of the present study was to identify and quantify associations between several endogenous (host) and exogenous (environmental) factors and immunoglobulin E (IgE) reactivity against Obsoletus complex-derived whole body extract or seven recombinant allergens, measured by ELISA. Data from 143 Icelandic horses and 177 Shetland ponies were analysed using multivariable models. In addition, the relationship between IgE reactivity and severity of clinical signs in IBH-affected horses was examined. Positive correlations were found between Obsoletus complex-specific IgE and severity of clinical signs. Disease status (IBH affected or control), breed and the interaction between IBH status and breed were significantly associated with IgE reactivity against several Obsoletus complex allergens. Significantly greater IgE reactivity was seen in IBH-affected horses compared to controls. The differences in IgE values between cases and controls were most pronounced in Icelandic horses. Shetland pony controls had significantly greater IgE reactivity compared to Icelandic horse controls, while differences in IgE values comparing Shetland pony cases and Icelandic horse cases were not significant. Severity of clinical signs and IgE reactivity in IBH-affected horses against several Obsoletus complex allergens appeared to be related. Consideration of the factors associated with Obsoletus complex-specific IgE in horses might further improve interpretation and accuracy of IgE ELISA test results within these breeds, although further research is required.
Estimation of hepatitis E virus (HEV) pig seroprevalence using Elisa and Western blot and comparison between human and pig HEV sequences in Belgium.
Thiry, D. ; Mauroy, A. ; Saegerman, C. ; Thomas, I. ; Wautier, M. ; Miry, C. ; Czaplicki, G. ; Berkvens, D. ; Praet, N. ; Poel, W.H.M. van der; Cariolet, R. ; Brochier, B. ; Thiry, E. - \ 2014
Veterinary Microbiology 172 (2014)3-4. - ISSN 0378-1135 - p. 407 - 414.
antibodies - swine - transmission - infection - netherlands - populations - prevalence - france - meat - farm
Zoonotic transmission of hepatitis E virus (HEV) is of special concern, particularly in high income countries were waterborne infections are less frequent than in developing countries. High HEV seroprevalences can be found in European pig populations. The aims of this study were to obtain prevalence data on HEV infection in swine in Belgium and to phylogenetically compare Belgian human HEV sequences with those obtained from swine. An ELISA screening prevalence of 73% (95% CI 68.8–77.5) was determined in Belgian pigs and a part of the results were re-evaluated by Western blot (WB). A receiver operating characteristic curve analysis was performed and scenarios varying the ELISA specificity relative to WB were analysed. The seroprevalences estimated by the different scenarios ranged between 69 and 81% and are in agreement with the high exposure of the European pig population to HEV. Pig HEV sequences were genetically compared to those detected in humans in Belgium and a predominance of genotype 3 subtype f was shown in both swine and humans. The high HEV seroprevalence in swine and the close phylogenetic relationships between pig and human HEV sequences further support the risk for zoonotic transmission of HEV between humans and pigs.
Schmallenberg virus detection in bovine semen after experimental infection of bulls.
Poel, W.H.M. van der; Parlevliet, J.M. ; Verstraten, E.R.A.M. ; Kooi, E.A. ; Hakze-van der Honing, R.W. van der; Stockhofe-Zurwieden, N. - \ 2014
Epidemiology and Infection 142 (2014)07. - ISSN 0950-2688 - p. 1495 - 1500.
antibodies - cattle
To study Schmallenberg virus (SBV) excretion in bovine semen after experimental infection, two bulls were inoculated subcutaneously with a SBV isolate (1 ml Vero cell culture 106 TCID50). After inoculation (at day 0), semen was collected daily from both animals for 21 days and samples were tested for SBV by qRT–PCR assay. At 24 days post-inoculation both animals were subjected to necropsy and the genital organs and lymph nodes draining these organs were also tested for SBV RNA (qRT–PCR). After SBV infection both animals in the study showed viraemia (qRT–PCR) with fever and diarrhoea. SBV RNA could be detected in semen from both animals. The highest SBV RNA concentrations in semen were found in the first week (days 4–7 post-inoculation) but concentrations were relatively low (Ct values 30–39). Viable SBV was only isolated from blood samples and not from semen or genital tissues.
Comparison of test methodologies for foot-and-mouth disease virus serotype A vaccine matching
Tekleghiorghis, T. ; Weerdmeester, K. ; Hemert-Kluitenberg, F. van; Moormann, R.J.M. ; Dekker, A. - \ 2014
Clinical and Vaccine Immunology 21 (2014)5. - ISSN 1556-6811 - p. 674 - 683.
binary ethylenimine - protection - potency - antibodies - cattle - elisa - variability - challenge - evolution - selection
Vaccination has been one of the most important interventions in disease prevention and control. The impact of vaccination largely depends on the quality and suitability of the chosen vaccine. To determine the suitability of a vaccine strain, antigenic matching is usually studied by in vitro analysis. In this study, we performed three in vitro test methods to determine which one gives the lowest variability and the highest discriminatory capacity. Binary ethylenimine inactivated vaccines, prepared from 10 different foot-and-mouth disease (FMD) virus serotype A strains, were used to vaccinate cattle (5 animals for each strain). The antibody titers in blood serum samples 3 weeks postvaccination (w.p.v.) were determined by a virus neutralization test, neutralization index test, and liquid-phase blocking enzyme-linked immunosorbent assay (ELISA). The titers were then used to calculate relationship coefficient (r1) values. These r1 values were compared to the genetic lineage using receiver operating characteristic (ROC) analysis. In the two neutralization test methods, the median titers observed against the test strains differed considerably, and the sera of the vaccinated animals did not always show the highest titers against their respective homologous virus strains. When the titers were corrected for test strain effect (scaling), the variability (standard error of the mean per vaccinated group) increased because the results were on a different scale, but the discriminatory capacity improved. An ROC analysis of the r1 value calculated on both observed and scaled titers showed that only r1 values of the liquid-phase blocking ELISA gave a consistent statistically significant result. Under the conditions of the present study, the liquid-phase blocking ELISA showed less variation and still had a higher discriminatory capacity than the other tests.
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