The double-antigen ELISA concept for early detection of Erns -specific classical swine fever virus antibodies and application as an accompanying test for differentiation of infected from marker vaccinated animals.

Emergency vaccination with live marker vaccines represents a promising control strategy for future classical swine fever (CSF) outbreaks, and the first live marker vaccine is available in Europe. Successful implementation is dependent on a reliable accompanying diagnostic assay that allows differentiation of infected from vaccinated animals (DIVA). As induction of a protective immune response relies on virus-neutralizing antibodies against E2 protein of CSF virus (CSFV), the most promising DIVA strategy is based on detection of Erns -specific antibodies in infected swine. The aim of this study was to develop and to evaluate a novel Erns -specific prototype ELISA (pigtype CSFV Erns Ab), which may be used for CSF diagnosis including application as an accompanying discriminatory test for CSFV marker vaccines. The concept of a double-antigen ELISA was shown to be a solid strategy to detect Erns -specific antibodies against CSFV isolates of different genotypes (sensitivity: 93.5%; specificity: 99.7%). Furthermore, detection of early seroconversion is advantageous compared with a frequently used CSFV E2 antibody ELISA. Clear differences in reactivity between sera taken from infected animals and animals vaccinated with various marker vaccines were observed. In combination with the marker vaccine CP7_E2alf, the novel ELISA represents a sensitivity of 90.2% and a specificity of 93.8%. However, cross-reactivity with antibodies against ruminant pestiviruses was observed. Interestingly, the majority of samples tested false-positive in other Erns -based antibody ELISAs were identified correctly by the novel prototype Erns ELISA and vice versa. In conclusion, the pigtype CSFV Erns Ab ELISA can contribute to an improvement in routine CSFV antibody screening, particularly for analysis of sera taken at an early time point after infection and is applicable as a DIVA assay. An additional Erns antibody assay is recommended for identification of false-positive results in a pig herd immunized with the licensed CP7_E2alf marker vaccine.

Expression of a truncated hepatitis E virus capsid protein in the protozoan organism Leishmania tarentolae and its application in a serological assay.

Zoonotic infections with hepatitis E virus (HEV) genotype 3 are presumably transmitted via contaminated pig meat products, which raises the necessity for enhanced serological surveillance of pig herds. The aim of the study was to set up a novel protein expression system to overcome the well-known problems in (HEV-) protein expression using the standard Escherichia coli tools such as inclusion body formation and loss of protein conformation. A recombinant strain of the protozoan organism Leishmania tarentolae (L. tarentolae) was therefore established. A fragment of HEV ORF2 coding for a truncated capsid protein of a porcine HEV strain was cloned and parts of the plasmid DNA were introduced into the Leishmania genome, resulting in stably transformed cells. Via a C-terminal His-tag the recombinant HEVΔORF2 protein could be purified and concentrated directly from the medium, resulting in a total protein amount of approximately 1.4 mg/l Leishmania culture. The recombinant protein was coated on ELISA plates and was proven to be highly reactive and well-suited to be applied in a serological assay. By investigating 144 porcine sera, the in-house assay detected specific antibodies in 43.1% of the samples and demonstrated a higher sensitivity than a commercially available antibody test. Taken together, it was shown that L. tarentolae exhibits a remarkable alternative expression strategy for viral antigens with considerable advantages of a eukaryotic protein expression host.

Involvement of coenzyme A esters and two new enzymes, an enoyl-CoA hydratase and a CoA-transferase, in the hydration of crotonobetaine to L-carnitine by Escherichia coli.

Two proteins (CaiB and CaiD) were found to catalyze the reversible biotransformation of crotonobetaine to L-carnitine in Escherichia coli in the presence of a cosubstrate (e.g., gamma-butyrobetainyl-CoA or crotonobetainyl-CoA). CaiB (45 kDa) and CaiD (27 kDa) were purified in two steps to electrophoretic homogeneity from overexpression strains. CaiB was identified as crotonobetainyl-CoA:carnitine CoA-transferase by MALDI-TOF mass spectrometry and enzymatic assays. The enzyme exhibits high cosubstrate specificity to CoA derivatives of trimethylammonium compounds. In particular, the N-terminus of CaiB shows significant identity with other CoA-transferases (e.g., FldA from Clostridium sporogenes, Frc from Oxalobacter formigenes, and BbsE from Thauera aromatica) and CoA-hydrolases (e.g., BaiF from Eubacterium sp.). CaiD was shown to be a crotonobetainyl-CoA hydratase using MALDI-TOF mass spectrometry and enzymatic assays. Besides crotonobetainyl-CoA CaiD is also able to hydrate crotonyl-CoA with a significantly lower Vmax (factor of 10(3)) but not crotonobetaine. The substrate specificity of CaiD and its homology to the crotonase confirm this enzyme as a new member of the crotonase superfamily. Concluding these results, it was verified that hydration of crotonobetaine to L-carnitine proceeds at the CoA level in two steps: the CaiD catalyzed hydration of crotonobetainyl-CoA to L-carnitinyl-CoA, followed by a CoA transfer from L-carnitinyl-CoA to crotonobetaine, catalyzed by CaiB. When gamma-butyrobetainyl-CoA was used as a cosubstrate (CoA donor), the first reaction is the CoA transfer. The optimal ratios of CaiB and CaiD during this hydration reaction, determined to be 4:1 when crotonobetainyl-CoA was used as cosubstrate and 5:1 when gamma-butyrobetainyl-CoA was used as cosubstrate, are different from that found for in vivo conditions (1:3).

Biotransformation of crotonobetaine to L(-)-carnitine in Proteus sp.

Two proteins, component I (CI) and component II (CII), catalyze the biotransformation of crotonobetaine to L(-)-carnitine in Proteus sp. CI was purified to electrophoretic homogeneity from cell-free extracts of Proteus sp. The N-terminal amino acid sequence of CI showed high similarity (80%) to the caiB gene product from Escherichia coli O44K74, which encodes the L(-)-carnitine dehydratase. CI alone was unable to convert crotonobetaine into L(-)-carnitine even in the presence of the cosubstrates crotonobetainyl-CoA or gamma-butyrobetainyl-CoA, which are essential for this biotransformation. The relative molecular mass of CI was determined to be 91.1 kDa. CI is composed of two identical subunits of molecular mass 43.6 kDa. The isoelectric point is 5.0. CII was purified to electrophoretic homogeneity from cell-free extracts of Proteus sp. and its N-terminal amino acid sequence showed high similarity (75%) to the caiD gene product of E. coli O44K74. The relative molecular mass of CII was shown to be 88.0 kDa, and CII is composed of three identical subunits of molecular mass 30.1 kDa. The isoelectric point of CII is 4.9. For the biotransformation of crotonobetaine to L(-)-carnitine, the presence of CI, CII, and a cosubstrate (crotonobetainyl-CoA or gamma-butyrobetainyl-CoA) were shown to be essential.

Epigenetic regulation of carnitine metabolising enzymes in Proteus sp. under aerobic conditions.

Proteus sp. is able to catalyse the reversible transformation of crotonobetaine into L(-)-carnitine during aerobic growth. Contrary to other Enterobacteriaceae no reduction of crotonobetaine into gamma-butyrobetaine could be detected in the culture supernatants. Activities of L(-)-carnitine dehydratase, carnitine racemasing system and crotonobetaine reductase could be determined enzymatically in cell-free extracts of Proteus sp. Small amounts of gamma-butyrobetaine were found in cell-free extracts, indicating that it accumulates in the cell and inhibits the crotonobetaine reductase. Crotonobetaine and L(-)-carnitine were able to induce enzymes of carnitine metabolism. gamma-Butyrobetaine and glucose repress carnitine metabolism in Proteus sp. Other betaines are neither inducers nor repressors. Monoclonal antibodies against purified CaiA from Escherichia coli O44K74 recognise an analogous protein in cell-free extract of Proteus sp. No cross-reactivity could be detected with monoclonal antibodies against purified CaiB and CaiD from E. coli O44K74.

In situ analysis of sulfur in the sulfur globules of phototrophic sulfur bacteria by X-ray absorption near edge spectroscopy.

During the oxidation of sulfide and thiosulfate purple and green sulfur bacteria accumulate globules of 'elemental' sulfur. Although essential for a thorough understanding of sulfur metabolism in these organisms, the exact chemical nature of the stored sulfur is still unclear. We applied sulfur K-edge X-ray absorption near edge spectroscopy (XANES) to probe the forms of sulfur in intact cells. Comparing XANES spectra of Allochromatium vinosum, Thiocapsa roseopersicina, Marichromatium purpuratum, Halorhodospira halophila and Chlorobium vibrioforme grown photolithoautotrophically on sulfide with reference probes (fingerprint method), we found sulfur chains with the structure R-S(n)-R. Evidence for the presence of sulfur rings, polythionates and anionic polysulfides in the sulfur globules of these bacteria was not obtained.