Serological diagnosis of bovine neosporosis: a Bayesian evaluation of two antibody ELISA tests for in vivo diagnosis in purchased and abortion cattle.

Currently, there are no perfect reference tests for the in vivo detection of Neospora caninum infection. Two commercial N caninum ELISA tests are currently used in Belgium for bovine sera (TEST A and TEST B). The goal of this study is to evaluate these tests used at their current cut-offs, with a no gold standard approach, for the test purpose of (1) demonstration of freedom of infection at purchase and (2) diagnosis in aborting cattle. Sera of two study populations, Abortion population (n=196) and Purchase population (n=514), were selected and tested with both ELISA's. Test results were entered in a Bayesian model with informative priors on population prevalences only (Scenario 1). As sensitivity analysis, two more models were used: one with informative priors on test diagnostic accuracy (Scenario 2) and one with all priors uninformative (Scenario 3). The accuracy parameters were estimated from the first model: diagnostic sensitivity (Test A: 93.54 per cent-Test B: 86.99 per cent) and specificity (Test A: 90.22 per cent-Test B: 90.15 per cent) were high and comparable (Bayesian P values >0.05). Based on predictive values in the two study populations, both tests were fit for purpose, despite an expected false negative fraction of ±0.5 per cent in the Purchase population and ±5 per cent in the Abortion population. In addition, a false positive fraction of ±3 per cent in the overall Purchase population and ±4 per cent in the overall Abortion population was found.

Follow-up of the Schmallenberg Virus Seroprevalence in Belgian Cattle.

Schmallenberg virus (SBV), which emerged in Northwestern Europe in 2011, is an arthropod-borne virus affecting primarily ruminants. Based on the results of two cross-sectional studies conducted in the Belgian ruminant population during winter 2011-2012, we concluded that at the end of 2011, almost the whole population had already been infected by SBV. A second cross-sectional serological study was conducted in the Belgian cattle population during winter 2012-2013 to examine the situation after the 2012 transmission period and to analyse the change in immunity after 1 year. A total of 7130 blood samples collected between 1st January and 28 February 2013 in 188 herds were tested for the presence of SBV-specific antibodies. All sampled herds tested positive and within-herd seroprevalence was estimated at 65.66% (95% CI: 62.28-69.04). A statistically significant decrease was observed between the beginning and the end of 2012. On the other hand, age-cohort-specific seroprevalence stayed stable from 1 year to the other. During winter 2012-2013, calves between 6 and 12 months had a seroprevalence of 20.59% (95% CI: 15.34-25.83), which seems to be an indication that SBV was still circulating at least in some parts of Belgium during summer-early autumn 2012. Results showed that the level of immunity against SBV of the animals infected has not decreased and remained high after 1 year and that the spread of the virus has slowed down considerably during 2012. This study also indicated that in the coming years, there are likely to be age cohorts of unprotected animals.

Large-scale cross-sectional serological survey of Schmallenberg virus in Belgian cattle at the end of the first vector season.

A cross-sectional survey was conducted in the Belgian cattle population after the first period of infection of the emerging Schmallenberg virus. A total number of 11 635 cattle from 422 herds sampled between 2 January and 7 March 2012 were tested for the presence of Schmallenberg-specific antibodies using an ELISA kit. Between-herd seroprevalence in cattle was estimated at 99.76% (95% CI: 98.34-99.97) and within-herd seroprevalence at 86.3% (95% CI: 84.75-87.71). An Intraclass Correlation Coefficient of 0.3 (P < 0.001) was found, indicating that the correlation between two animals within a herd with respect to their serological status was high. Those results corroborate the conclusion that the Schmallenberg virus was widespread in Belgium during winter 2011. Seroprevalence was shown to be statistically associated to the animal's age (P < 0.0001): with 64.9% (95% CI: 61.34-68.3) estimated for the 6-12 months of age, 86.79% (95% CI: 84.43-88.85) for the 12-24 months of age and 94.4% (95% CI: 93.14-95.44) for the animals older than 24 months. Based on the results of the described serological survey, we can conclude that after the first Schmallenberg virus episode, almost every Belgian cattle has already been in contact with the virus. In consequence, the vast majority of the host animals should have developed post infection protective immunity against the virus.

Survival capacity in water of Arcobacter species under different temperature conditions.

AIMS: To assess the survival capacity in vitro of arcobacters in water at temperatures applied in the food industry. METHODS AND RESULTS: Four strains of each Arcobacter species were inoculated in potable water and water with 1% organic material and stored at 4, 7, 20, 52, 56 and 60 degrees C. Samples were taken at known time points and the numbers of bacteria were determined on Arcobacter-selective medium. All Arcobacter species remained viable for a temperature-dependent period of time, although Arcobacter butzleri displayed a significant longer survival and heat resistance. No significant intraspecies differences were detected, resulting in no definite identification of origin or strain dependency. The survival period for all species was prolonged in the presence of the organic material only for the low temperatures. CONCLUSIONS: The present study demonstrates that water can act as a reservoir and as a potential source of Arcobacter contamination to humans and animals. SIGNIFICANCE AND IMPACT OF THE STUDY: This study assessed for the first time the survival of all human-related Arcobacter species in water. Particularly A. butzleri showed to be the most robust species with regard to temperature which is interesting as that species is often found in human clinical specimens.

Discrepancy between the occurrence of Arcobacter in chickens and broiler carcass contamination.

Both Campylobacter and Arcobacter are commonly present on broiler carcasses. For Campylobacter, the superficial contamination originates predominantly from fecal contamination during slaughter. In contrast with Campylobacter, the source of the Arcobacter contamination is not clear. In several studies, arcobacters have been isolated in poultry processing plants from the carcasses and slaughter equipment, but not from the intestinal content. In literature, contradictory reports about the Arcobacter colonization of the chicken gut have been published. In most of those studies, arcobacters were not isolated from cecal content nor from litter or the feathers, though some studies reported the isolation of arcobacters from cloacal swab samples. The present study assessed if arcobacters are part of the chicken intestine, skin, or feather flora. Because no isolation protocol has been validated for poultry intestinal content, a previously developed Arcobacter isolation procedure for feces from livestock animals was first validated. With this method, a good repeatability, in-lab reproducibility and sensitivity, and a good suppression of the chicken fecal accompanying flora were achieved when 125 mg/L of 5-fluorouracil, 10 mg/L of amphotericine B, 100 mg/L of cycloheximide, 16 mg/L of cefoperazone, 64 mg/L of novobiocine, and 64 mg/L of trimethoprim were applied. The validated method was used to examine the presence of arcobacters in and on living chickens of 4 flocks at slaughter age. Because arcobacters were not isolated from the intestinal tract nor from the skin or feathers of the birds, this study was not able to identify arcobacters as part of the intestinal or skin flora, nor could confirm the role of process water as reservoir. However, the results clearly demonstrated that the time period for processing the samples and the way of sample collection are crucial in the interpretation of epidemiological studies. As the reservoir of the carcass contamination remains unidentified, studies about the capacity of arcobacters to colonize the chicken intestinal tract may contribute in the assessment of the transmission routes of this emerging foodborn pathogen.

Crystallization of helix pomatia agglutinin (HPA), a protein from the edible snail. erratum

One of the authors was omitted in the published version of the paper by Lisgarten et al. (1999) Acta Cryst. D55, 1903-1905. The full author list is given above.

Crystallization of Helix pomatia agglutinin (HPA), a protein from the edible snail.

Crystals of Helix pomatia agglutinin (HPA) have been grown by the hanging-drop technique using polyethylene glycol as the precipitant at 293 K. Over a period of one to two weeks the crystals grew to maximum dimensions of 0.10 x 0.05 x 0.02 mm. The crystals belong to space group P6(3)22, with unit-cell dimensions a = b = 63.3, c = 105. 2 A and Z = 12 identical monomers of M(r) = 13 kDa, aggregating into two 78 kDa hexameric protein molecules per unit cell, each with symmetry 32 (D(3)). The diffraction pattern extends to 3.6 A at 293 K.

The use of non-immune plasma powder in the prophylaxis of neonatal Escherichia coli diarrhoea in calves.

The protective use of plasma powder from cattle and swine against experimentally induced neonatal E. coli diarrhoea in colostrum-deprived calves was examined. Diarrhoea was induced with a strain expressing F5+ fimbriae and a strain expressing F17+ fimbriae. In all groups supplemented with bovine plasma powder, diarrhoea and fever were less severe than in the control groups. For the groups infected with the F5+ E. coli strain, a reduction in excretion of the challenge strain by 2-4 orders of magnitude and by 1-2 orders of magnitude was seen when supplemented with bovine plasma powder at a dose of 25 g/l milk and 10 g/l milk, respectively. The bovine plasma powder showed also beneficial effects in the F17+ infected groups. No mortality, no septicaemia and no severe clinical signs were observed. Concerning the excretion of the E. coli F17+ strain in the faeces, no significant difference with the control group was found. Swine plasma powder showed little beneficial effect on E. coli diarrhoea in calves in this study.

Protection of just weaned pigs against infection with F18+ Escherichia coli by non-immune plasma powder.

The anti-colonization effect of porcine plasma powder against experimentally induced postweaning diarrhoea and oedema disease in just weaned piglets was examined. Piglets were infected with an Escherichia coli strain expressing F18ac fimbriae and producing SLTIIv- and LT-toxins. Reduced fecal excretion of the challenge strain and protection against clinical symptoms was obtained by daily supplementation of the feed with either 90 or 45 g of plasma powder. However, the piglets receiving 90 g of plasma powder a day showed diarrhoea and reduced weight gain compared to the piglets receiving 45 g of plasma powder a day. The diarrhoea was attributed to biogenic amines released from excessive protein in the diet.

Pig heart fumarase contains two distinct substrate-binding sites differing in affinity.

A eukaryotic fumarase is for the first time unequivocally shown to contain two distinct substrate-binding sites. Pig heart fumarase is a tetrameric enzyme consisting of four identical subunits of 50 kDa each. Besides the true substrates L-malate and fumarate, the active sites (sites A) also bind their analogs D-malate and oxaloacetate, as well as the competitive inhibitor glycine. The additional binding sites (sites B) on the other hand also bind the substrates and their analogs D-malate and oxaloacetate, as well as L-aspartate which is not an inhibitor. Depending on the pH, the affinity of sites B for ligands (Kd being in the millimolar range) is 1-2 orders of magnitude lower than the affinity of sites A (of which Kd is in the micromolar range). However, saturating sites B results in an increase in the overall activity of the enzyme. The benzenetetracarboxyl compound pyromellitic acid displays very special properties. One molecule of this ligand is indeed able to bind into a site A and a site B at the same time. Four molecules of pyromellitic acid were found to bind per molecule fumarase, and the affinity of the enzyme for this ligand is very high (Kd = 0.6 to 2.2 microM, depending on the pH). Experiments with this ligand turned out to be crucial in order to explain the results obtained. An essential tyrosine residue is found to be located in site A, whereas an essential methionine residue resides in or near site B. Upon limited proteolysis, a peptide of about 4 kDa is initially removed, probably at the C-terminal side; this degradation results in inactivation of the enzyme. Small local conformational changes in the enzyme are picked up by circular dichroism measurements in the near-UV region. This spectrum is built up of two tryptophanyl triplets, the first one of which is modified upon saturating the active sites (A), and the second one upon saturating the low affinity binding sites (B).

Role of Mg2+ in the structure and activity of maize (Zea mays L.) isocitrate lyase: indications for hysteretic behaviour.

The role of Mg2+ in the structure and activity of maize isocitrate lyase has been studied by CD, limited proteolysis, protection by ligands against inactivation, and activity measurements at various metal concentrations. From CD and trypsinolysis experiments, the existence of high-affinity binding sites for Mg2+ was demonstrated, and a KdME of 200 microM was determined. Both free enzyme (E) and enzyme molecules with high-affinity sites occupied (ME) are catalytically competent, the former showing 40% of the activity of the latter. Mg2+ thus acts as a non-essential activator. A second Mg2+-binding site with a KdMEM of 6 mM was revealed from protection experiments by increasing Mg2+ concentrations against inactivation. From activity measurements at different Mg2+ concentrations, the affinity of the enzyme for the Mg2+-isocitrate complex (MI) was determined to be KdE(MI) = 9 microM. Maize isocitrate lyase was shown to display hysteretic behaviour. Filling the low-affinity binding sites with Mg2+ induces a conformational change in the high-affinity binding sites resulting in an even higher affinity for Mg2+ (KdME* = 40 microM). On lowering the Mg2+ concentration again, the enzyme only responds slowly: the time needed for all enzyme molecules to return to the conformation at which KdME is 200 microM was found to be 60 min. Finally it was shown that the high-affinity binding site for Mg2+ is not formed at low (4 degrees C) temperature.

Chicken egg yolk antibodies against F18ab fimbriae of Escherichia coli inhibit shedding of F18 positive E. coli by experimentally infected pigs.

F18ab and F18ac are antigenic variants of a colonizing fimbria commonly found on E. coli associated with postweaning diarrhea and edema disease in pigs. Chicken F18ab antibodies were obtained by immunising hens with purified F18ab fimbriae. For their in vitro characterisation antibodies were isolated from diluted egg yolks by ammonium sulfate precipitation. In vitro adhesion tests demonstrated that the chicken F18ab antibodies inhibited attachment of F18ab positive E. coli bacteria to the intestinal mucosa. Just weaned piglets were experimentally infected with an F18ab positive edema disease strain of E. coli, or with an F18ac positive postweaning diarrhea E. coli strain. The animals were infected on the second day of a period during which chicken F18ab antibodies were added to their feed. During the same period, pigs of the control group received commercial eggs in which no F18 antibodies were detected. In both experimental infections the excretion of the F18 positive strain was reduced in pigs that received the F18ab antibodies as compared to the control animals. The F18ab antibodies diminished the cases of diarrhea and death in animals infected with F18ac positive E. coli.

Trypanosome-binding proteins of the tsetse flies Glossina palpalis gambiensis and G. morsitans morsitans.

In this paper we describe a new, selective approach to identify protein ligand-receptor interactions between an arthropod vector and the parasite it transmits. Biotinylated vector proteins were incubated with living parasites in physiological conditions. After extensive washing, the parasites were subjected to SDS-PAGE electrophoresis and the polypeptides were electroblotted onto nitrocellulose membrane. Staining with avidin-horseradish peroxidase revealed only biotin-labeled proteins from the vector which were bound to the parasite. A multitude of tissue-specific proteins of Glossina palpalis gambiensis and G. morsitans morsitans proteins, able to bind to cultured procyclic trypanosomes of Trypanosoma brucei spp., has been demonstrated. The relevance of these interactions in relation to the developmental journey of the trypanosome in the tsetse fly is briefly discussed.

Ligand binding on to maize (Zea mays) malate synthase: a structural study.

A kinetic and ligand binding study on maize (Zea mays) malate synthase is presented. It is concluded from kinetic measurements that the enzyme proceeds through a ternary-complex mechanism. Michaelis constants (Km,glyoxylate and Km,acetyl-CoA) were determined to be 104 microM and 20 microM respectively. C.d. measurements in the near u.v.-region indicate that a conformational change is induced in the enzyme by its substrate, glyoxylate. From these studies we are able to calculate the affinity for the substrate (Kd,glyoxylate) as 100 microM. A number of inhibitors apparently trigger the same conformational change in the enzyme, i.e. pyruvate, glycollate and fluoroacetate. Another series of inhibitors bearing more bulky groups and/or an extra carboxylic acid also induce a conformational change, which is, however, clearly different from the former one. Limited proteolysis with trypsin results in cleavage of malate synthase into two fragments of respectively 45 and 19 kDa. Even when no more intact malate synthase chains are present, the final enzymic activity still amounts to 30% of the original activity. If trypsinolysis is performed in the presence of acetyl-CoA, the cleavage reaction is appreciably slowed down. The dissociation constant for acetyl-CoA (Kd,acetyl-CoA) was calculated to be 14.8 microM when the glyoxylate subsite is fully occupied by pyruvate and 950 microM (= 50 x Km) when the second subsite is empty. It is concluded that malate synthase follows a compulsory-order mechanism, glyoxylate being the first-binding substrate. Glyoxylate triggers a conformational change in the enzyme and, as a consequence, the correctly shaped binding site for acetyl-CoA is created. Demetallization of malate synthase has no effect on the c.d. spectrum in the near u.v.-region. Moreover, glyoxylate induces the same spectral change in the absence of Mg2+ as in its presence. Nevertheless, malate synthase shows no activity in the absence of the cation. We conclude that Mg2+ is essential for catalysis, rather than for the structure of the enzyme's catalytic site.

A specific association between the glyoxylic-acid-cycle enzymes isocitrate lyase and malate synthase.

There is accumulating evidence that metabolic pathways are organized in vivo as multienzyme clusters or metabolons. To assess interactions between consecutive enzymes of a pathway in vitro, it is usually essential to modify the physical properties of water around the enzymes, e.g. by immobilizing the latter onto a solid support. Such immobilized enzyme preparations can be embedded in agarose gels and used for affinity electrophoresis [Beeckmans, S., Van Driessche, E. & Kanarek, L. (1989) Eur. J. Biochem. 183, 449-454; Beeckmans, S., Van Driessche, E. & Kanarek, L. (1990) J. Cell. Biochem. 43, 297-306]. In this study we use the aforementioned technique to investigate the association between two plant glyoxylic acid cycle enzymes, i.e. isocitrate lyase and malate synthase. A specific histochemical staining technique is described for both enzymes. Affinity electrophoresis using either isocitrate lyase or malate synthase as the immobilized enzyme clearly shows that associations are formed between both enzymes. Moreover, experiments with metabolically unrelated enzymes prove that the observed interaction is specific.

Tissue treatment for whole mount internal lectin staining in the nematodes Caenorhabditis elegans, Panagrolaimus superbus and Acrobeloides maximus.

Four different fixation schemes, using ten fluorescent-labelled lectins, were investigated for whole mount internal staining of three rhabditid nematodes: Caenorhabditis elegans, Panagrolaimus superbus and Acrobeloides maximus. Acetone-only fixation was found to give strong and reproducible staining, which could be prevented either by periodate treatment of the organisms or by specific inhibitory sugars of the lectins under investigation. Whereas the use of either phosphate or TRIS buffers had no effect on the staining pattern or the fluorescence intensity, the incubation time as well as the incubation temperature affected the staining reaction. The best results were obtained upon overnight incubation at 4 degrees C: the lectin staining could be inhibited in all cases, except for the intestinal brush border of C. elegans by the lectin of Lens culinaris.

Purification of the glyoxylate cycle enzyme malate synthase from maize (Zea mays L.) and characterization of a proteolytic fragment.

A purification scheme is described for the glyoxylate cycle enzyme malate synthase from maize scutella. With our procedure, large amounts of extremely pure enzyme can easily be prepared. Purification involves a heat denaturation step, followed by ammonium sulfate precipitation, and chromatography on DEAE-cellulose and Blue Dextran-Sepharose. Catalase and malate dehydrogenase, which are the most persistent contaminants, are completely removed by this procedure. Maize malate synthase is an octameric protein with a subunit molecular weight of 64 kDa. Purity of the enzyme preparation was demonstrated by SDS-polyacrylamide gel electrophoresis and by isoelectric focusing (pI = 5.0). Pure malate synthase can be stored without appreciable loss of activity at -70 degrees C in 200 mM Hepes buffer containing 6 mM MgCl2 and 2 mM 2-mercaptoethanol, pH 7.6. Maize malate synthase contains no covalently linked carbohydrate residues. The enzyme requires Mg2+ ions for activity. From circular dichroism measurements we estimate that the secondary structure of the enzyme consists of 30% alpha-helical and almost no (5%) beta-pleated sheet segments. A 45-kDa polypeptide, which contaminates malate synthase preparations if the purification starts from seedlings older than 2.5 days, is shown to be a degradation product of malate synthase. Together with full-length chains, these 45-kDa polypeptides are able to take part in octameric oligomer formation.

Immobilized enzymes as tools for the demonstration of metabolon formation. A short overview.

In recent years it has become clear that a cell cannot be visualized as a 'bag' filled with enzymes dissolved in bulk water. The aqueous-phase properties in the interior of a cell are, indeed, essentially different from those of an ordinary aqueous solution. Large amounts of water are believed to be organized in layers at the surface of intracellular structural proteins and membranes. Such considerations prompt us to reconsider the operation and regulation of metabolic pathways. Enzymes of metabolic pathways are nowadays thought to be clustered and operate as 'metabolons'. Very often interactions between enzymes of a pathway can exclusively be evidenced in vitro in media which are known to reduce the water concentration in the vicinity of the proteins. Immobilized enzyme preparations have been shown to be excellent tools for this type of research. We describe here some recent studies where immobilized enzymes have been used in various applications to investigate associations among enzymes of a number of different metabolic pathways (glycolysis/gluconeogenesis, citric acid cycle and its connection to the electron transport chain, aspartate-malate shuttle, glyoxylate cycle). Advantages and disadvantages of the different techniques are also discussed.

Inhibition of adhesion of enterotoxigenic Escherichia coli cells expressing F17 fimbriae to small intestinal mucus and brush-border membranes of young calves.

Enterotoxigenic Escherichia coli strains expressing F17 fimbriae bind to the intestinal mucosa of young calves. F17 fimbriae recognize receptors present in the mucus layer and the brush-border membranes from duodenum, jejunum and ileum. The adhesion of E. coli F17 can be inhibited by several glycoproteins. Adhesion is also inhibited by pretreatment of mucus and brush-border membranes with sodium metaperiodate. The use of glycoconjugates as potential adhesion-blockers is further discussed.

The purification and physicochemical characterization of maize (Zea mays L.) isocitrate lyase.

A purification scheme is described for the glyoxylate cycle enzyme isocitrate lyase from maize scutella. Purification involves an acetone precipitation and a heat denaturation step, followed by ammonium sulfate precipitation and chromatography on DEAE-cellulose and on blue-Sepharose. The latter step results in the removal of the remaining malate dehydrogenase activity, and of a high molecular mass (62 kDa) but inactive degradation product of isocitrate lyase. Catalase can be completely removed by performing the DEAE-cellulose chromatography in the presence of Triton X-100. Pure isocitrate lyase can be stored without appreciable loss of activity at -70 degrees C in 5 mM triethanolamine buffer containing 6 mM MgCl2, 7 mM 2-mercaptoethanol, and 50% (v/v) glycerol, pH 7.6. Maize isocitrate lyase is a tetrameric protein with a subunit molecular mass of 64 kDa. Purity of the enzyme preparation was demonstrated by polyacrylamide gel electrophoresis in the presence of dodecylsulfate, in acid (pH 3.2) urea and by isoelectric focusing (pI = 5.1). Maize isocitrate lyase is devoid of covalently linked sugar residues. From circular dichroism measurements we estimate that its structure comprises 30% alpha-helical and 15% beta-pleated sheet segments. The enzyme requires Mg2+ ions for activity, and only Mn2+ apparently is able to replace this cation to a certain extent. The kinetics of the isocitrate lyase-catalyzed cleavage reaction were investigated, and the amino acid composition of the maize enzyme was determined. Finally the occurrence of an association between maize isocitrate lyase and catalase was observed. Such a multienzyme complex may be postulated to play a protective role in vivo.