Christian Seyboldt

The Gene 10 (UL49.5) Product of Equine Herpesvirus 1 Is Necessary and Sufficient for Functional Processing of Glycoprotein M

ABSTRACT The functional cooperation of equine herpesvirus 1 (EHV-1) glycoprotein M (gM) and the gene 10 (UL49.5) product was analyzed. Transient-transfection experiments using gM and UL49.5 expression plasmids as well as RK13 cell lines constitutively expressing UL49.5 (RK49.5) or gM (RKgM) demonstrated that the endo-β-N-acetylglucosaminidase H (endo H)-resistant mature form of gM was detectable only after coexpression of the two proteins. Deletion of the EHV-1 UL49.5-homologous gene 10 in strain KyA resulted in a small-plaque phenotype and up to 190-fold-reduced virus titers. The growth defects of the mutant KyAΔ49.5 virus, which were very similar to those of a gM-negative KyA virus, could be completely compensated for by growth of the mutant virus on RK49.5 cells or by repairing the deletion of gene 10 in the revertant virus KyAΔ49.5R. Analysis of cells infected with the UL49.5-negative EHV-1 demonstrated that gM was not transported to the trans-Golgi network in the absence of the UL49.5 product. In contrast, gM was efficiently transported and processed to the endo H-resistant mature form in KyAΔ49.5-infected RK49.5 cells. Furthermore, radioimmunoprecipitation experiments demonstrated that gM maturation was observed only if a 10,000-M r protein was coprecipitated with gM in KyA- or KyAΔ49.5R-infected cells or virions. This protein was absent in cells infected with KyΔ49.5 or KyAΔgM, suggesting that it was the EHV-1 UL49.5 product. Taken together, our results demonstrate that the expression of the EHV-1 UL49.5 product is necessary and sufficient for gM processing and that it is required for efficient virus replication.

Clostridium difficile Genotypes in Piglet Populations in Germany

ABSTRACT Clostridium difficile was isolated from 147 of 201 (73%) rectal swabs of piglets from 15 farms of Lower Saxony and North Rhine-Westphalia. In 14 farms, 14 to 100% (mean, 78%) of the animals tested were culture positive. The rate of isolation was 68% postpartum, increased to 94% in animals 2 to 14 days of age, and declined to 0% for animals 49 days of age and older. There was no link between isolation and antibiotic treatment or diarrhea of piglets. Strains were assigned to 10 PCR ribotypes, and up to 4 PCR ribotypes were found to be present at the same time on a farm. The closely related PCR ribotypes 078 (55%) and 126 (20%) were most frequently recovered and were present in 13 of the 14 positive farms. The comparison of multilocus VNTR (variable number of tandem repeats) analysis (MLVA) data from this study and previously published data on human, porcine, and bovine PCR ribotype 078 isolates from 5 European countries revealed genetic differences between strains of different geographic origin and confirmed the relatedness of human and porcine C. difficile isolates. This study demonstrated that the human-pathogenic PCR ribotypes 078 and 126 are predominant in piglets in Germany. The results suggest that presence of C. difficile is correlated with animal age but not with antibiotic treatment or clinical disease. MLVA indicated that strains of the same geographical origin are often genetically related and corroborated the hypothesis of a close epidemiological connection between human and porcine C. difficile isolates.

Presence of Clostridium difficile PCR ribotype clusters related to 033, 078 and 045 in diarrhoeic calves in Germany.

This study provides data on the distribution and relationship of C. difficile PCR ribotypes in diarrhoeic calves in Germany. C. difficile was isolated from 176 of 999 (17.6 %) faecal samples or swabs of diarrhoeic calves from 603 farms collected between January 2010 and August 2012 by eight federal laboratories of six states. Strains were assigned to 17 PCR ribotypes. PCR ribotypes 033 (57 %), 078 (17 %) and 045/FLI01 (closest match to 045 in the WEBRIBO database; 9 %) were found the most frequently. Nine per cent of all culture-positive tested animals shed more than one multiple locus variable number tandem repeat analysis (MLVA) or PCR ribotype. Eight PCR ribotypes with related profiles (including 033, 078 and 045/FLI01) representing 92 % of all isolates were grouped into three clusters. Molecular relatedness was supported by the absence of the MLVA locus A6Cd only in clustered strains and identical toxin gene profiles for strains within each cluster. Previously reported mulitilocus sequence typing analysis for PCR ribotypes that were also recovered in this study found identical sequence types and a tcdC deletion (Δ39 bp) for 033, 045, 078 and 126 (ST-11), confirming this clustering. A different geographical occurrence of PCR ribotypes was shown for cluster 033 (found more frequently in southern Germany) and 045 (found more frequently in northern Germany). This study showed that clusters of C. difficile PCR ribotypes related to 033, 078 and 045 are predominant in diarrhoeic calves in Germany. The high number of strains belonging to PCR ribotype 078 demonstrated that diarrhoeic calves are also potential reservoirs for human pathogenic C. difficile strains.

Deletion of gene 52 encoding glycoprotein M of equine herpesvirus type 1 strain RacH results in increased immunogenicity

The immunogenicity of equine herpesvirus type 1 (EHV-1) strain RacH was compared to a RacH virus in which gene 52 encoding glycoprotein M (gM) was interrupted by insertion of LacZ (HDeltagM-Ins) and a RacH with 75% of gene 52 was deleted and replaced by LacZ (HDeltagM-HS). HDeltagM-Ins failed to produce full-length gM, but the carboxy-terminal portion was still expressed. No gM expression was detected in HDeltagM-HS-infected cells. Mice were immunised once with 1x10(3) to 1x10(5) plaque-forming units (PFU) of RacH or mutant viruses and challenged with virulent RacL11 virus 29 days later. A dose-dependence of protection was observed in RacH-immunised mice, and following immunisation with 1x10(4) or 1x10(3) PFU body weight losses and increased virus titres in lungs were observed after challenge infection. HDeltagM-HS-immunised mice were completely protected even after immunisation with 1x10(3) PFU. Mice immunised with 1x10(3) PFU of HDeltagM-Ins but not the higher doses showed signs of disease after challenge infection.

Development and validation of a multiplex real-time PCR for detection of Clostridium chauvoei and Clostridium septicum

Clostridium chauvoei is the causative agent of blackleg in cattle and sheep. The clinical symptoms of this severe disease are very similar to that of malignant edema (Clostridium septicum), infections of other Clostridium species belonging to the gas edema complex, and anthrax (Bacillus anthracis). C. chauvoei and C. septicum are closely related taxa and share many phenotypic properties hampering diagnosis by using traditional microbiological methods. Thus, there is a need for a fast and reliable identification method for specific detection of both species in clinical samples. The multiplex real-time PCR assay presented here is based on the detection of the spo0A gene and enables the simultaneous identification of C. chauvoei and C. septicum. The assay design includes an amplification control DNA template for the recognition of PCR-inhibitors. Assay validation was performed using a collection of 29 C. chauvoei, 38 C. septicum strains and 26 strains of other Clostridium species. Furthermore, the real-time PCR assay was successfully tested on tissue samples from 19 clinical blackleg cases. The assay allowed the reliable detection of one picogram DNA which represents approximate 239 genome equivalents.

DNA Microarray-Based PCR Ribotyping of Clostridium difficile

ABSTRACT This study presents a DNA microarray-based assay for fast and simple PCR ribotyping of Clostridium difficile strains. Hybridization probes were designed to query the modularly structured intergenic spacer region (ISR), which is also the template for conventional and PCR ribotyping with subsequent capillary gel electrophoresis (seq-PCR) ribotyping. The probes were derived from sequences available in GenBank as well as from theoretical ISR module combinations. A database of reference hybridization patterns was set up from a collection of 142 well-characterized C. difficile isolates representing 48 seq-PCR ribotypes. The reference hybridization patterns calculated by the arithmetic mean were compared using a similarity matrix analysis. The 48 investigated seq-PCR ribotypes revealed 27 array profiles that were clearly distinguishable. The most frequent human-pathogenic ribotypes 001, 014/020, 027, and 078/126 were discriminated by the microarray. C. difficile strains related to 078/126 (033, 045/FLI01, 078, 126, 126/FLI01, 413, 413/FLI01, 598, 620, 652, and 660) and 014/020 (014, 020, and 449) showed similar hybridization patterns, confirming their genetic relatedness, which was previously reported. A panel of 50 C. difficile field isolates was tested by seq-PCR ribotyping and the DNA microarray-based assay in parallel. Taking into account that the current version of the microarray does not discriminate some closely related seq-PCR ribotypes, all isolates were typed correctly. Moreover, seq-PCR ribotypes without reference profiles available in the database (ribotype 009 and 5 new types) were correctly recognized as new ribotypes, confirming the performance and expansion potential of the microarray.

Clostridium spp. discrimination with a simple bead-based fluorescence assay

C. chauvoei is the causative agent of blackleg, an endogenous bacterial infection which usually affects cattle and other ruminants. Due to the fact that the symptoms of this severe disease are very similar to the phenotype caused by an infection with C. septicum, a reliable differentiation of C. chauvoei from other Clostridium spp. is mandatory. Traditional microbiological detection methods are time consuming and the proper specification is hampered by the overgrowing tendency of swarming C. septicum colonies when both species are in the clinical sample. Thus, there is an urgent need to improve and simplify the specific detection of C. chauvoei and C. septicum. We report an easy and fast Clostridium spp. discrimination method via a magnetic bead-based fluorescence assay. To that end, the target DNA was amplified using 16S-23S rDNA spacer region specific primers. These PCR products were employed to generate single-stranded capture probe DNA, which was immobilized on magnetic beads. Functionalized magnetic particles exhibit numerous advantages, like their simple manipulation in combination with a huge binding capacity of biomolecules and make therefore excellent biosensors. In this context, the discrimination between C. chauvoei and C. septicum was realized by means of hybridization with complementary detection probe DNA. Finally, fluorescence spectroscopy allowed the signal readout. With this approach a precise discrimination between C. chauvoei, C. septicum and C. carnis was accomplished.

Dry-reagent-based PCR as a novel tool for the rapid detection of Clostridium spp.

Improved conventional PCR techniques are required for the rapid on-site detection of human and animal diseases. In this context, a PCR method using dry-stored reagents intended for the detection of Clostridium spp. is presented. Basic PCR reagents (BSA, PCR buffer, MgCl₂ and primers), which were dried on polyolefin matrices, showed stability at ambient temperatures for up to 10 months without any loss of functionality. An outstanding advantage of our amelioration is the elimination of PCR process errors caused by the improper storage and handling of liquid reagents. Moreover, our PCR-based amplification can be performed in less than 30 min, saving time compared with conventional detection methods. Thus, dry-reagent-based PCR is implementable in a suitcase-like modular device for the rapid on-site detection of microbial pathogens such as blackleg of ruminants caused by Clostridium chauvoei.

First report of two complete Clostridium chauvoei genome sequences and detailed in silico genome analysis

Clostridium (C.) chauvoei is a Gram-positive, spore forming, anaerobic bacterium. It causes black leg in ruminants, a typically fatal histotoxic myonecrosis. High quality circular genome sequences were generated for the C. chauvoei type strain DSM 7528T (ATCC 10092T) and a field strain 12S0467 isolated in Germany. The origin of replication (oriC) was comparable to that of Bacillus subtilis in structure with two regions containing DnaA boxes. Similar prophages were identified in the genomes of both C. chauvoei strains which also harbored hemolysin and bacterial spore formation genes. A CRISPR type I-B system with limited variations in the repeat number was identified. Sporulation and germination process related genes were homologous to that of the Clostridia cluster I group but novel variations for regulatory genes were identified indicative for strain specific control of regulatory events. Phylogenomics showed a higher relatedness to C. septicum than to other so far sequenced genomes of species belonging to the genus Clostridium. Comparative genome analysis of three C. chauvoei circular genome sequences revealed the presence of few inversions and translocations in locally collinear blocks (LCBs). The species genome also shows a large number of genes involved in proteolysis, genes for glycosyl hydrolases and metal iron transportation genes which are presumably involved in virulence and survival in the host. Three conserved flagellar genes (fliC) were identified in each of the circular genomes. In conclusion this is the first comparative analysis of circular genomes for the species C. chauvoei, enabling insights into genome composition and virulence factor variation.

The zoonotic potential of Clostridium difficile from small companion animals and their owners

Background Clostridium difficile infections (CDI) in humans range from asymptomatic carriage to life-threatening intestinal disease. Findings on C. difficile in various animal species and an overlap in ribotypes (RTs) suggest potential zoonotic transmission. However, the impact of animals for human CDI remains unclear. Methods In a large-scale survey we collected 1,447 fecal samples to determine the occurrence of C. difficile in small companion animals (dogs and cats) and their owners and to assess potential epidemiological links within the community. The Germany-wide survey was conducted from July 2012-August 2013. PCR ribotyping, Multilocus VNTR Analysis (MLVA) and PCR detection of toxin genes were used to characterize isolated C. difficile strains. A database was defined and logistic regression used to identify putative factors associated with fecal shedding of C. difficile. Results In total, 1,418 samples met the inclusion criteria. The isolation rates for small companion animals and their owners within the community were similarly low with 3.0% (25/840) and 2.9% (17/578), respectively. PCR ribotyping revealed eight and twelve different RTs in animals and humans, respectively, whereas three RTs were isolated in both, humans and animals. RT 014/0, a well-known human hospital-associated lineage, was predominantly detected in animal samples. Moreover, the potentially highly pathogenic RTs 027 and 078 were isolated from dogs. Even though, C. difficile did not occur simultaneously in animals and humans sharing the same household. The results of the epidemiological analysis of factors associated with fecal shedding of C. difficile support the hypothesis of a zoonotic potential. Conclusions Molecular characterization and epidemiological analysis revealed that the zoonotic risk for C. difficile associated with dogs and cats within the community is low but cannot be excluded.