Immune cell kinetics in the ovine abomasal mucosa following hyperimmunization and challenge with Haemonchus contortus

Sheep were sensitized by repeated infection with Haemonchus contortus L3, followed by a 12 week rest period, and an abomasal cannula was surgically implanted in all sheep. Seven of the sensitized sheep were subsequently challenged with 50 000 H. contortus L3 while 4 control sheep were challenged with saline. Biopsy samples were taken using a fibreoptic endoscope on days 0, 1, 2, 3, 5, 7 and 28 after challenge and leukocyte subpopulations quantified by (immuno)histology. Differential blood cell counts were performed on the same days. At the end of the trial, sheep showed significantly reduced worm burdens compared to unsensitized control sheep, confirming their resistance status. Both blood and tissue eosinophils, as well as tissue γδ TCR⁺ cells were rapidly elevated by day 1 post L3 challenge (pc), peaking at day 3 pc. There was a slight increase in tissue CD4 T cells at day 2 pc, peaking at day 3 pc while no significant changes in CD8 T cells were observed. B cells (CD45R⁺) increased later into challenged tissues with a peak at 5 days pc. All tissue lymphocyte subpopulations as well as tissue and blood eosinophils were reduced by day 7 pc before increasing again at day 28 pc, suggesting separate responses to larval and adult antigens. In contrast, globule leukocytes and mucosal mast cells only showed one peak at day 5 pc and 28 pc, respectively. Unexpectedly, globule leukocytes correlated significantly with tissue eosinophils but not mucosal mast cells. The results are consistent with an early eosinophil-mediated killing of L3, possibly recruited by IL-5 produced by γδ T cells. In contrast to post-mortem studies, abomasal cannulation allowed sequential analysis of both early and late time points in the same animal, providing a more complete picture of cellular interactions at both peripheral and local sites, and their correlation with the different stages of parasite development.     

Quantitative verification of the correspondence between the Welfare Quality® operational definition of farm animal welfare and the opinion of Flemish farmers,citizens and vegetarians

The Welfare Quality® project has developed an operational definition of farm animal welfare, which consists of 12 criteria grouped into four principles (‘good feeding’, ‘good housing’, ‘good health’ and ‘appropriate behaviour’). As part of a larger survey, 204 farmers, 72 vegetarians, and a representative sample of 459 adult citizens in Flanders (Belgium) scored the importance of each of the 12 criteria on a 1–10 scale, and divided 100 points across the four principles according to the perceived relative weights for farm animal welfare. These data were analysed to verify quantitatively whether (1) the grouping of 12 criteria into the four principles, and (2) the relative weights used by Welfare Quality® for aggregating the 12 criteria into an overall welfare judgement correspond with the perception of these three stakeholder groups from a region and setting not previously covered by Welfare Quality® studies. Moreover, the effects of various socio-demographic variables on the importance allocated to the welfare criteria and principles were investigated. Vegetarians and females gave consistently higher importance scores to all 12 criteria as compared with farmers, citizens and males, respectively, particularly for the (criteria belonging to the) principle ‘appropriate behaviour’. Farmers allocated more weight to ‘good feeding’ than did vegetarians or citizens. These differences confirm that farmers view animal welfare more in terms of biological functioning (instead of affective states or natural living) as compared to other citizens, and vegetarians in particular. As the concept has a different meaning to different people, any operational definition of animal welfare is unlikely to match equally well with the perception of every single stakeholder group or person. The structure of the Welfare Quality® operational definition with the 12 criteria aggregated into four principles, corresponds well with the opinion of the stakeholders as they judged all 12 criteria as rather important to extremely important for farm animal welfare. Factor analysis restricted to contain four factors confirmed the grouping of the criteria into the correct principles. Exploratory factor analysis using Eigenvalues > 1 as extraction criterion indicated, however, that from the citizen perspective two principles (‘good feeding & health’ and ‘appropriate behaviour & housing’) rather than four would suffice. The main discrepancy with the Welfare Quality® approach concerns the aggregation procedure: the stakeholders did not consider all four principles as equally important, and the relative importance allocated to criteria corresponded poorly across principles. Some of these discrepancies were consistent across the three different stakeholder groups.     

A comparison of fixatives suitable for scanning electron microscopy of Habronema spp

Members of the genus Habronema (Nematoda: Habronematidae) were preserved in 4 fixatives for examination with scanning electron microscopy (SEM). Tegumental features of the anterior and posterior extremities of these specimens were compared to evaluate the effect of the fixatives: modified Flemming's solution, glutaraldehyde (GA), Karnovsky's fixative and 70% ethanol. Fixatives were assessed on the appearance of the tegument, evidence of any wrinkling, shrinkage or swelling and the degree of extension in the male tail. Seventy per cent ethanol gave the most satisfactory results.     

Detection of residues of the coccidiostat diclazuril in poultry tissues by liquid chromatography-tandem mass spectrometry after withdrawal of medicated feed

A liquid chromatography-tandem mass spectrometric (LC-MS/MS) method for the quantitative determination of diclazuril in poultry tissues and feed is presented. A simple clean up with an organic solvent was carried out. A reversed-phase C(18) column was used for the high-performance liquid chromatography (HPLC) to separate the analyte with a gradient of acetonitrile and water as mobile phase. The precursor ions produced by electrospray negative ionization were selected for collisional dissociation. Validation of the methods was performed based on Commission Decision 2002/657/EC (Off. J. Eur. Communities 2002, L221, 8-36). For the detection of diclazuril in poultry meat, the decision limit was found to be 0.5 microg/kg. An animal experiment was set up in which 70 chickens were held for 6 weeks. From day 22 until day 32, they were fed feed containing 730 microg/kg diclazuril. From day 33 until day 42, every day six chickens were slaughtered, and breast, thigh, and liver were analyzed. Average steady-state concentrations of 94, 135, and 722 microg/kg in breast, thigh, and liver were obtained, respectively. Nine days after withdrawal of the medicated feed, diclazuril was still present in the different sample types.     

Cells, cytokines and other molecules associated with rejection of gastrointestinal nematode parasites

Infections with gastrointestinal nematodes represent a major problem for human health and animal health and production. The physical size of the parasite, its changing life-cycle stages and the relative inaccessibility to host cells pose unique challenges to the immune system, which has evolved specialized strategies for parasite control. This paper reviews the work performed in the authors' laboratories to identify components that are involved in the natural rejection response against ruminant gastrointestinal nematode parasites, in particular Haemonchus contortus in sheep. The results of these studies indicate that stage-specific antibodies act in concert with effector cells, in particular globular leukocytes (intraepithelial mast cells) and eosinophils, appropriately activated/primed by type 2 (T2) cytokines, to initiate different mechanisms of parasite expulsion and killing. In addition, other molecules, in particular carbohydrate binding galectins, may be involved in strengthening the final effector phase of the rejection response.     

An electron microscopic study of intra-erythrocytic stages of Babesia bovis in the brain capillaries of infected splenectomized calves.

Splenectomized vaccine donor calves undergoing primary reactions to Babesia bovis infections may develop cerebral babesiosis which leads to death if not treated in time. A brain biopsy was performed on an artificially-infected animal showing nervous symptoms and the tissue was immediately processed for electron microscopic examination. Virtually every erythrocyte in the brain capillaries sectioned was infected with B. bovis. Intra-erythrocytic merozoites, trophozoites and dividing trophozoites were indentified. Important features of the piriform merozoites included a reduced apical complex consisting of the anterior polar ring, microtubules, rhoptries and micronemes. Unidentified membrane-bound bodies, mostly spherical in shape, were observed anterior to the nucleus. The trophozoites showed very little structural differentiation and no food vacuoles or micropores could be detected. Each trophozoite produced 2 identical merozoites and the parent cell became totally incorporated in the daughter merozoites in the multiplication process. Projections were seen radiating from the surface of infected erythrocytes which appeared to adhere to other surfaces on contact. This probably resulted in the sludging of infected erythrocytes in the capillaries. The latter observations coincide with those described for Babesia argentina.     

Do citizens and farmers interpret the concept of farm animal welfare differently?

Animal welfare is of increasing importance in livestock production and consumption debates. However, discordance exists between citizens' and farmers' perception of animal welfare. Since the search for, and the realization of improvements towards farm animal welfare is strongly driven by citizen expectations, it is of utmost importance to better understand this perceptual discordance. A quantitative study was done in Flanders, Belgium during 2006, including citizens as well as farmers, to obtain a detailed insight in the way the multi-dimensional concept of farm animal welfare is valued. This allowed to discriminate between issues of agreement and disagreement. In general, a similar interpretation of farm animal welfare in terms of animal welfare related aspect's ranking was found. Differences were mainly related to aspects dealing with the ability to engage in natural behaviour on the one hand and with production process-related aspects on the other hand. Citizens evaluate the current state of animal welfare as rather problematic, while farmers report a more satisfactory evaluation of the present condition of farm animal welfare. Especially differing opinions regarding the ability to engage in natural behaviour, together with aspects related to pain, stress and the availability of space seem to contribute to the discordance between farmers and citizens in terms of evaluative beliefs.     

The possibility of positive selection for both F18(+)Escherichia coli and stress resistant pigs opens new perspectives for pig breeding

F18(+)Escherichia coli infections causing post-weaning diarrhoea and/or oedema disease are a major cause of economic losses in pig industry. To date, no preventive strategy can protect pigs from F18(+)E. coli infections. One of the most attractive approaches to eliminate F18(+)E. coli infections is the selection for pigs that are resistant to F18(+)E. coli infections. However, this strategy was not believed to be favourable because of reports of genetic association with the stress-susceptibility gene in the Swiss Landrace. To investigate this potential association more thoroughly, 131 randomly selected Belgian hybrid pigs were genotyped for both the F18(+)E. coli resistance alleles (FUT1(A)) and the stress-susceptibility alleles (RYR1(T)) and their association was investigated by determining the linkage disequilibrium. This linkage disequilibrium (LD=-0.0149) is close to zero and does not differ significantly from 0 (likelihood ratio test chi(1)(2)=1.123, P=0.29), demonstrating no association between the FUT1(A) and RYR1(T) alleles. Furthermore, only a small fraction (4.6%) of the Belgian pigs was found to be resistant to F18(+)E. coli infections. Our results suggest that selection for F18(+)E. coli resistant pigs might be an attractive approach to prevent pigs from F18(+)E. coli infections, unlike to what has previously been postulated.     

A universal microarray detection method for identification of multiple Phytophthora spp. using padlock probes

The genus Phytophthora consists of many species that cause important diseases in ornamental, agronomic, and forest ecosystems worldwide. Molecular methods have been developed for detection and identification of one or several species of Phytophthora in single or multiplex reactions. In this article, we describe a padlock probe (PLP)-based multiplex method of detection and identification for many Phytophthora spp. simultaneously. A generic TaqMan polymerase chain reaction assay, which detects all known Phytophthora spp., is conducted first, followed by a species-specific PLP ligation. A 96-well-based microarray platform with colorimetric readout is used to detect and identify the different Phytophthora spp. PLPs are long oligonucleotides containing target complementary sequence regions at both their 5' and 3' ends which can be ligated on the target into a circular molecule. The ligation is point mutation specific; therefore, closely related sequences can be differentiated. This circular molecule can then be detected on a microarray. We developed 23 PLPs to economically important Phytophthora spp. based upon internal transcribed spacer-1 sequence differences between individual Phytophthora spp. Tests on genomic DNA of many Phytophthora isolates and DNA from environmental samples showed the specificity and utility of PLPs for Phytophthora diagnostics.     

Acid-induced cold gelation of globular proteins: effects of protein aggregate characteristics and disulfide bonding on rheological properties

The process of cold gelation of ovalbumin and the properties of the resulting cold-set gels were compared to those of whey protein isolate. Under the chosen heating conditions, most protein was organized in aggregates. For both protein preparations, the aggregates consisted of covalently linked monomers. Both types of protein aggregates had comparable numbers of thiol groups exposed at their surfaces but had clearly different shapes. During acid-induced gelation, the characteristic ordering caused by the repulsive character disappeared and was replaced by a random distribution. This process did not depend on aggregate characteristics and probably applies to any type of protein aggregate. Covalent bonds are the main determinants of the gel hardness. The formation of additional disulfide bonds during gelation depended on the number and accessibility of thiol groups and disulfide bonds in the molecule and was found to clearly differ between the proteins studied. However, upon blocking of the thiol groups, long fibrillar structures of ovalbumin contribute significantly to gel hardness, demonstrating the importance of aggregate shape.