Theodoor Niewold

Selection and optimization of proteolytically stable llama single-domain antibody fragments for oral immunotherapy

We previously demonstrated that oral application of the recombinant single-domain antibody fragment (VHH) clone K609, directed against Escherichia coli F4 fimbriae, reduced E. coli-induced diarrhoea in piglets, but only at high VHH doses. We have now shown that a large portion of the orally applied K609 VHH is proteolytically degraded in the stomach. Stringent selection for proteolytic stability identified seven VHHs with 7- to 138-fold increased stability after in vitro incubation in gastric fluid. By DNA shuffling we obtained four clones with a further 1.5- to 3-fold increased in vitro stability. These VHHs differed by at most ten amino acid residues from each other and K609 that were scattered over the VHH sequence and did not overlap with predicted protease cleavage sites. The most stable clone, K922, retained 41% activity after incubation in gastric fluid and 90% in jejunal fluid. Oral application of K922 to piglets confirmed its improved proteolytic stability. In addition, K922 bound to F4 fimbriae with higher affinity and inhibited fimbrial adhesion at lower VHH concentrations. K922 is thus a promising candidate for prevention of piglet diarrhoea. Furthermore, our findings could guide selection and improvement by genetic engineering of other recombinant antibody fragments for oral use.

The effect of dietary spray-dried porcine plasma on clinical response in weaned piglets challenged with a pathogenic Escherichia coli

Weaned piglets were used to determine the effect of dietary spray-dried porcine plasma (SDPP) on the clinical response to an infection with a pathogenic Escherichia coli (E. coli) O139:K82 LT(-). The piglets were divided into two groups of 10 animals each. One group was fed the control diet containing soybean(meal) plus whey powder. The test piglets were fed a diet with 8% SDPP. Piglets were orally infected with the challenge strain on days 6 and 7 after weaning. The experimental period lasted 14 days after which the piglets were euthanised and necropsied. Faecal samples were collected daily for bacteriological analysis. Segments of jejunum, caecum and rectum were removed for bacteriological analysis post mortem. Feed intake and weight gain, faecal and condition scores and body temperature were measured daily. In the control and SDPP groups, 6 and 7 piglets died from diarrhoea. The average daily feed intake (ADFI) and average daily gain (ADG) were substantially higher in the SDPP group than in the control group. SDPP-fed piglets generally had a more favourable faecal score and a healthier appearance than did the control piglets. The faecal excretion of E. coli O139:K82 was similar for control and test piglets. There were no diet effects on the E. coli O139:K82 counts at different sites of the intestine. In this experiment, the inclusion of SDPP at an economically acceptable percentage in the diet could not prevent piglet losses due to challenge with a pathogenic E. coli, but improvements of ADG, ADFI and faecal and condition scores were achieved.

Casein related amyloid, characterization of a new and unique amyloid protein isolated from bovine corpora amylacea

Amyloid bodies can be found in mammary secretory tissue of various species. These corpora amylacea (CA) have a lamellated structure, contain amyloid fibrils and are predominantly located in the alveolar lumina. The nature of the amyloid was not known, but CA were suggested to originate either from milk casein or mammary alveolar epithelial keratin. In the present report, bovine CA were analyzed histochemically. Furthermore, CA were isolated, analyzed and the amyloid was purified and characterized by amino acid sequencing. CA amyloid appeared to be potassium permanganate sensitive and tryptophan positive, and in this respect different from most other amyloid types except for AA and beta-2 microglobulin amyloid. Gel filtration of purified amyloid fibrils showed a HMW peak and a major 4 kD peak. N-terminal amino acid sequencing showed the amyloid to consist of tryptic-like peptides with an unusually high content of amino acids with bulky side chains. The amyloid protein was identified as derived from alpha-S2-casein. The fragments are of varying length (32, 33 and 45 amino acids), but all start at position 81 of alpha-S2-casein. We have identified a new and unique amyloid protein, and we propose to designate it as A alpha-S2C according to the guidelines for amyloid nomenclature.

A review of porcine pathophysiology: a different approach to disease.

Abstract Diseases are often thought to result from a single cause. Although this is sometimes the case, e.g. with a highly virulent infection such as Classical Swine Fever (CSF), more often clinical disease in swine herds results from multiple predisposing factors. This is especially true in modern intensive pig husbandry, in which the role of highly infectious diseases is limited to (nonetheless devastating) outbreaks. More important nowadays are diseases, although associated with an agent, without a clear pathogenesis. The emphasisin disease control thusfar has been on treatment, eradication and prevention. This has been achieved by focusing attention on husbandry factors, such as climate, housing, hygiene, management, and nutrition. Although this approach has been successful for a number of diseases, several health problems are persistent. There are strong indications that in the latter, intrinsic animal factors are important. Successful handling of these problems requires knowledge of the (patho)physiology of the pig. In this article, several characteristics of pig physiology associated with the occurrence of disease are described. It appears that the modern (fattening) pig is exceptional among other animal species in that its cardiovascular system is mismatched to its body weight. It is argued that this particular disposition causes relatively minor disturbances to have major consequences in the pig. This concept of pig physiology is central to the understanding of the hitherto poorly understood pathogenesis of several diseases, such as oedema disease.

Serum amyloid A3 (SAA3), not SAA1 appears to be the major acute phase SAA isoform in the pig.

The acute-phase serum amyloid A (SAA) protein family comprises two main circulating (systemic) isoforms, SAA1 and SAA2, synthesised in liver and one local isoform, SAA3, produced in extrahepatic tissues. Systemic and local SAA show structural differences, which suggests different functions. In the pig, AA-amyloidosis is extremely uncommon, and the structural protein in swine has characteristics of systemic SAA. The only pig SAA sequences published so far, either derived form hepatic or extrahepatic sites have been designated SAA2, but the translated protein shows the properties of SAA3 proteins. The aim of this study was to characterise all the porcine SAA isoforms by sequencing from cDNA and genomic DNA obtained form multiple porcine tissues. Primer pairs were designed to amplify presumably all isoforms of SAA firstly and then specifically for each isotype. Results show that the only isotype isolated and sequenced both from hepatic and extrahepatic tissues correspond to a SAA3-like amino acid sequence. No SAA1-like sequences were identified, which could be indicative of the gene being very rare and consistent with the observed resistance to AA-amyloidosis. Finally, it is concluded that the pig is unique among other species in that the main circulating hepatic SAA isotype shows the characteristics of local highly alkaline SAA. This likely precludes a function as apolipoprotein.

Transcriptomics of enterotoxigenic Escherichia coli infection. Individual variation in intestinal gene expression correlates with intestinal function

Acute secretory diarrhea is a major cause of morbidity and mortality in young animals and humans. Deaths result from excessive fluid and electrolyte losses. The disease is caused by non-invasive bacteria such as Vibrio cholerae and Escherichia coli which produce enterotoxins, however, much less is known about the role of individual host responses. Here we report the response of intact porcine small intestinal mucosa to infection with enterotoxigenic E. coli (ETEC). Jejunal segments in four piglets were infused with or without ETEC, and perfused for 8h, and net absorption measured. Microarray analysis at 8h post-infection showed significant differential regulation of on average fifteen transcripts in mucosa, with considerable individual variation. Differential net absorption varied between animals, and correlated negatively with the number of up regulated genes, and with one individual gene (THO complex 4). This shows that quantitative differences in gene regulation can be functionally linked to the physiological response in these four animals.

Summary of the first round analyses of the Third International Workshop on Swine Leukocyte Differentiation Antigens

The reactivity of 155 monoclonal antibodies submitted to the Third International Workshop on Swine Leukocyte Differentiation Antigens, together with 41 internal standards, was analysed by flow cytometry on 29 different pig cell targets as well as two human cell targets as a means of establishing suitable panels of monoclonal antibodies for more detailed clustering analyses by the various subsections of the workshop. Results were collected either without further gating, with gating based on FS/SS characteristics or with gating based on the co-expression of a reference antibody in two-colour flow cytometry. The CD or SWC reactivity of the internal standards had been established in previous workshops. Data sets were subsequently analysed by statistical clustering using the Leucocyte Typing Database IV software. The resulting 18 cluster groups were allocated to the appropriate second round sections of the workshop, after reviewing the overall cellular reactivity of each cluster as well as the specificity of known standards which clustered in a group.

E. coli heat labile toxin (LT) inactivation by specific polyphenols is aggregation dependent.

Recently, polyphenol extracts were suggested to inhibit binding of Escherichia coli heat labile enterotoxin (LT) to its intestinal receptor GM1. Therefore, polyphenols are promising feed or food supplements to combat enterotoxigenic infections. Little is known of the precise mechanism, or the type of polyphenol required. Here, seven different polyphenols were tested in vitro (1) for inhibition of LT binding to GM1 (GM1-ELISA), (2) for LT inhibitory activity in the cAMP Vero-cell assay, and (3) by testing the aggregating properties of polyphenols with LT using molecular weight exclusion membrane filters, and by centrifugation techniques. Results showed only three out of seven polyphenols, pentagalloylglucose (PGG), epigallocatechingallate (EGCG) and gallocatechingallate (GCG), to effectively inhibit binding of LT to GM1, and to inhibit induction of cAMP in Vero cells, and that PGG is the most effective. Blocking of the GM1 receptor is unlikely as a mechanism because pre-incubation of GM1 with polyphenols had no effect. Co-incubation of polyphenols with forskolin did not interfere with cAMP production in Vero cells, showing that polyphenol activity is not directly related to cAMP. It is concluded that the inhibitory activities of these three polyphenols may coincide with the formation of large (>100 kDa) LT-polyphenol aggregates. Enterotoxin inactivation appears to require a minimum of two galloyl moieties in polyphenol structure and the pentagalloyl PGG is the most effective.

Disease incidence and immunological traits for the selection of healthy pigs: A review

Summary Disease is a major issue in animal production systems and society demands that the use of medicines and vaccines be reduced. This review describes the breeding approaches that could be used to improve disease resistance and focuses especially on their application to pigs. Disease reduction by genetic means has certain advantages through cumulative and permanent effects, and direct and indirect selection methods are available. Direct selection for disease incidence requires, besides a unique pig identification and disease registration system, challenge routines that are inconvenient in intensive pig production. Indirect selection for the expression of immune capacity may be an alternative but requires detailed knowledge of the different components of the immune system. There is ample opportunity for genetic improvement of the immune capacity because immune traits show substantial genetic variation between pigs. We therefore conclude that indirect selection via immune traits is very interesting, also for practical implementation, and that there is an urgent need for knowledge, within lines, about the genetic relationships between immune capacity traits and resistance to specific diseases or to disease incidence in general. Furthermore, knowledge about the relationship between immune system traits and production traits is needed as well as knowledge about the effect of selection on the epidemiology of disease at a farm/population level and on the host‐pathogen interaction and coevolution.

Relationship between wheat characteristics and nutrient digestibility in broilers: comparison between total collection and marker (titanium dioxide) technique

Three wheat cultivars (Orpheus, Rustic, and Viscount) were used to formulate 3 test feeds (62.4% wheat) in a broiler digestibility trial. The diets were fed to male Ross 308 broiler chickens. The wheat cultivars mainly differed in their amount of non-starch polysaccharides ( NSP: ). The cultivar Orpheus was chosen to represent a high amount of NSP (102 g/kg DM), whereas the cultivars Rustic and Viscount represented low amounts of NSP (83.4 g/kg DM and 73.9 g/kg DM, respectively). Furthermore, the cultivars Orpheus and Viscount were feed quality wheat, whereas Rustic was a milling quality wheat. Nutrient digestibilities and AMEn contents of the diets were measured from 18 to 22-days-old by total excreta collection, or with the use of the indigestible marker titanium dioxide. In addition, the ileal viscosity was measured when the broilers were 25-days-old. Wheat cultivar affected N retention, DM digestibility, and AMEn. In general, the feed formulated with the high NSP wheat cultivar Orpheus resulted in the least favorable nutrient digestibilities and AMEn, whereas the results were better when the feed was formulated with the low NSP cultivars Viscount and Rustic. Feeding the Rustic cultivar caused the highest intestinal viscosity, although this was not reflected in the animal responses. Nutrient digestibilities and AMEn content of the diets were lower when calculated with the titanium dioxide marker than with the total excreta collection procedure. Moreover, the P-values of the effect of wheat cultivar on DM digestibility, N retention, crude fat digestibility and AMEn were lower with the use of the titanium dioxide marker. It can be concluded that wheat cultivar affected nutrient digestibility and AMEn, and that the observed differences were related to the amount of NSP. Furthermore, both the titanium dioxide marker and the total excreta collection methods showed the same trends despite the different values obtained. The titanium dioxide marker method was the simplest to use and produced valid results.