Metabolic Traits of Bovine Shiga Toxin-Producing Escherichia Coli (STEC) Strains with Different Colonization Properties.

Cattle harbor Shiga toxin-producing Escherichia coli (STEC) in their intestinal tract, thereby providing these microorganisms with an ecological niche, but without this colonization leading to any clinical signs. In a preceding study, genotypic characterization of bovine STEC isolates unveiled that their ability to colonize cattle persistently (STECper) or only sporadically (STECspo) is more closely associated with the overall composition of the accessory rather than the core genome. However, the colonization pattern could not be unequivocally linked to the possession of classical virulence genes. This study aimed at assessing, therefore, if the presence of certain phenotypic traits in the strains determines their colonization pattern and if these can be traced back to distinctive genetic features. STECspo strains produced significantly more biofilm than STECper when incubated at lower temperatures. Key substrates, the metabolism of which showed a significant association with colonization type, were glyoxylic acid and L-rhamnose, which were utilized by STECspo, but not or only by some STECper. Genomic sequences of the respective glc and rha operons contained mutations and frameshifts in uptake and/or regulatory genes, particularly in STECper. These findings suggest that STECspo conserved features leveraging survival in the environment, whereas the acquisition of a persistent colonization phenotype in the cattle reservoir was accompanied by the loss of metabolic properties and genomic mutations in the underlying genetic pathways.

Effect of vitamin E supplementation in milk replacer and Shiga toxoid vaccination on serum α-tocopherol, performance, haematology and blood chemistry in male Holstein calves.

Vitamin E (vit E), an essential antioxidant for maintaining the stability of biological membranes and the function of the immune system, is considered to support adaptive immune responses and performance in cattle. The principal virulence factor of Shiga toxin (Stx)-producing Escherichia coli (STEC), the eponymous Stx, modulates cellular immune responses in cattle, the primary STEC reservoir. Active and passive immunization of calves with Shiga toxoids (rStxMUT ) was recently shown to reduce the STEC shedding. Here, we examined the influence of vit E on calves' serum α-tocopherol, performance, haematology, blood chemistry and its interaction with rStxMUT immunization. Data from calves having received passive (colostrum from immunized cows) and active (intramuscularly at 5th and 8th weeks of life) vaccination with rStxMUT (n = 24) were compared to unvaccinated controls (n = 24; fed with low anti-Stx colostrum, placebo injected). For each vaccination group, data were analysed according to the level of vit E supplementation offered by milk replacer (188 IU all-rac-α-tocopheryl acetate daily [VitEM ] vs. 354 IU [VitEH ]). An increase by 79% in daily vit E supplementation led to slightly higher serum α-tocopherol level and earlier concentrate intake at the beginning of the experiment without significant differences in live weight gain, haematology, blood chemistry parameters and peripheral CD4+ and CD8+ T-cell subpopulations. rStxMUT vaccination modulated the CD4+ /CD8+ ratio irrespective of vit E supplementation but decreased concentrate intake in VitEH in a time-dependent manner. Results of our study indicate that an increase in daily vit E supplementation vastly fails to exert effects on laboratory parameters and growth performance. However, observed interactive effects of vit E supply and vaccination on the regulation of feed intake deserves further attention.

Decreased STEC shedding by cattle following passive and active vaccination based on recombinant Escherichia coli Shiga toxoids.

The principal virulence factor of Shiga toxin (Stx)-producing Escherichia coli (STEC), the eponymous Stx, modulates cellular immune responses in cattle, the primary STEC reservoir. We examined whether immunization with genetically inactivated recombinant Shiga toxoids (rStx1MUT/rStx2MUT) influences STEC shedding in a calf cohort. A group of 24 calves was passively (colostrum from immunized cows) and actively (intra-muscularly at 5th and 8th week) vaccinated. Twenty-four calves served as unvaccinated controls (fed with low anti-Stx colostrum, placebo injected). Each group was divided according to the vitamin E concentration they received by milk replacer (moderate and high supplemented). The effective transfer of Stx-neutralizing antibodies from dams to calves via colostrum was confirmed by Vero cell assay. Serum antibody titers in calves differed significantly between the vaccinated and the control group until the 16th week of life. Using the expression of activation marker CD25 on CD4+CD45RO+ cells and CD8αhiCD45RO+ cells as flow cytometry based read-out, cells from vaccinated animals responded more pronounced than those of control calves to lysates of STEC and E. coli strains isolated from the farm as well as to rStx2MUT in the 16th week. Summarized for the entire observation period, less fecal samples from vaccinated calves were stx1 and/or stx2 positive than samples from control animals when calves were fed a moderate amount of vitamin E. This study provides first evidence, that transfer to and induction in young calves of Stx-neutralizing antibodies by Shiga toxoid vaccination offers the opportunity to reduce the incidence of stx-positive fecal samples in a calf cohort.

Complete Annotated Genome Sequences of Two Shiga Toxin-Producing Escherichia coli Strains and One Atypical Enteropathogenic E. coli Strain, Isolated from Naturally Colonized Cattle of German Origin.

Shiga toxin-producing Escherichia coli (STEC) strains are important zoonotic enteric pathogens with the main reservoir in cattle. Here, we present the genomes of two STEC strains and one atypical enteropathogenic E. coli strain from cattle origin, obtained during a longitudinal study in German cattle herds.

Evaluation of applicability of DNA microarray-based characterization of bovine Shiga toxin-producing Escherichia coli isolates using whole genome sequence analysis.

We assessed the ability of a commercial DNA microarray to characterize bovine Shiga toxin-producing Escherichia coli (STEC) isolates and evaluated the results using in silico hybridization of the microarray probes within whole genome sequencing scaffolds. From a total of 69,954 reactions (393 probes with 178 isolates), 68,706 (98.2%) gave identical results by DNA microarray and in silico probe hybridization. Results were more congruent when detecting the genoserotype (209 differing results from 19,758 in total; 1.1%) or antimicrobial resistance genes (AMRGs; 141 of 26,878; 0.5%) than when detecting virulence-associated genes (VAGs; 876 of 22,072; 4.0%). Owing to the limited coverage of O-antigens by the microarray, only 37.2% of the isolates could be genoserotyped. However, the microarray proved suitable to rapidly screen bovine STEC strains for the occurrence of high numbers of VAGs and AMRGs and is suitable for molecular surveillance workflows.

Evidence for Contemporary Switching of the O-Antigen Gene Cluster between Shiga Toxin-Producing Escherichia coli Strains Colonizing Cattle.

Shiga toxin-producing Escherichia coli (STEC) comprise a group of zoonotic enteric pathogens with ruminants, especially cattle, as the main reservoir. O-antigens are instrumental for host colonization and bacterial niche adaptation. They are highly immunogenic and, therefore, targeted by the adaptive immune system. The O-antigen is one of the most diverse bacterial cell constituents and variation not only exists between different bacterial species, but also between individual isolates/strains within a single species. We recently identified STEC persistently infecting cattle and belonging to the different serotypes O156:H25 (n = 21) and O182:H25 (n = 15) that were of the MLST sequence types ST300 or ST688. These STs differ by a single nucleotide in purA only. Fitness-, virulence-associated genome regions, and CRISPR/CAS (clustered regularly interspaced short palindromic repeats/CRISPR associated sequence) arrays of these STEC O156:H25 and O182:H25 isolates were highly similar, and identical genomic integration sites for the stx converting bacteriophages and the core LEE, identical Shiga toxin converting bacteriophage genes for stx1a, identical complete LEE loci, and identical sets of chemotaxis and flagellar genes were identified. In contrast to this genomic similarity, the nucleotide sequences of the O-antigen gene cluster (O-AGC) regions between galF and gnd and very few flanking genes differed fundamentally and were specific for the respective serotype. Sporadic aEPEC O156:H8 isolates (n = 5) were isolated in temporal and spatial proximity. While the O-AGC and the corresponding 5' and 3' flanking regions of these aEPEC isolates were identical to the respective region in the STEC O156:H25 isolates, the core genome, the virulence associated genome regions and the CRISPR/CAS elements differed profoundly. Our cumulative epidemiological and molecular data suggests a recent switch of the O-AGC between isolates with O156:H8 strains having served as DNA donors. Such O-antigen switches can affect the evaluation of a strain's pathogenic and virulence potential, suggesting that NGS methods might lead to a more reliable risk assessment.

The Accessory Genome of Shiga Toxin-Producing Escherichia coli Defines a Persistent Colonization Type in Cattle.

UNLABELLED: Shiga toxin-producing Escherichia coli (STEC) strains can colonize cattle for several months and may, thus, serve as gene reservoirs for the genesis of highly virulent zoonotic enterohemorrhagic E. coli (EHEC). Attempts to reduce the human risk for acquiring EHEC infections should include strategies to control such STEC strains persisting in cattle. We therefore aimed to identify genetic patterns associated with the STEC colonization type in the bovine host. We included 88 persistent colonizing STEC (STEC(per)) (shedding for ≥4 months) and 74 sporadically colonizing STEC (STEC(spo)) (shedding for ≤2 months) isolates from cattle and 16 bovine STEC isolates with unknown colonization types. Genoserotypes and multilocus sequence types (MLSTs) were determined, and the isolates were probed with a DNA microarray for virulence-associated genes (VAGs). All STEC(per) isolates belonged to only four genoserotypes (O26:H11, O156:H25, O165:H25, O182:H25), which formed three genetic clusters (ST21/396/1705, ST300/688, ST119). In contrast, STEC(spo) isolates were scattered among 28 genoserotypes and 30 MLSTs, with O157:H7 (ST11) and O6:H49 (ST1079) being the most prevalent. The microarray analysis identified 139 unique gene patterns that clustered with the genoserotypes and MLSTs of the strains. While the STEC(per) isolates possessed heterogeneous phylogenetic backgrounds, the accessory genome clustered these isolates together, separating them from the STEC(spo) isolates. Given the vast genetic heterogeneity of bovine STEC strains, defining the genetic patterns distinguishing STEC(per) from STEC(spo) isolates will facilitate the targeted design of new intervention strategies to counteract these zoonotic pathogens at the farm level. IMPORTANCE: Ruminants, especially cattle, are sources of food-borne infections by Shiga toxin-producing Escherichia coli (STEC) in humans. Some STEC strains persist in cattle for longer periods of time, while others are detected only sporadically. Persisting strains can serve as gene reservoirs that supply E. coli with virulence factors, thereby generating new outbreak strains. Attempts to reduce the human risk for acquiring STEC infections should therefore include strategies to control such persisting STEC strains. By analyzing representative genes of their core and accessory genomes, we show that bovine STEC with a persistent colonization type emerged independently from sporadically colonizing isolates and evolved in parallel evolutionary branches. However, persistent colonizing strains share similar sets of accessory genes. Defining the genetic patterns that distinguish persistent from sporadically colonizing STEC isolates will facilitate the targeted design of new intervention strategies to counteract these zoonotic pathogens at the farm level.

Instructive even after a decade: Complete results of initial virological diagnostics and re-evaluation of molecular data in the German rabies virus "outbreak" caused by transplantations.

In 2005, six patients in Germany received solid organs and both corneas from a donor with an unrecognized rabies infection. Initial virological diagnostics with the machinery available at the two national reference laboratories could quickly clarify the situation. Rabies virus antigen was detected in the organ donor's brain. In two of the three recipients with neurological alterations, intra vitam diagnosis was achieved by conventional RT-PCRs. Comparison of the phylogenetic relatedness of the different viral isolates proved transmission from the donor and, consequently, also established the diagnosis for the third patient. As indicated by the titre of neutralizing antibodies, the liver transplant recipient was protected from the lethal infection due to a vaccination against rabies virus, which he had received more than 15 years ago. All samples from the recipients of the corneas were invariably negative. Re-evaluation of the molecular data by real-time PCR did not lead to an improvement of intra vitam diagnosis but provided intriguing insights regarding the relative amounts of rabies virus RNA in different body fluids and peripheral organs. In saliva and skin, they were 250-200,000 times lower than in the infected patient's brains. Furthermore, in saliva samples taken serially from the same patient fluctuations by a factor of 160-500 were recorded. These findings highlight the problems of intra vitam diagnosis of rabies virus infections and make understandable why the virus can escape from all diagnostic attempts. Finally, in this context one should recall an almost trivial fact: Simple and appropriate postexposure prophylaxis could not only have saved the young organ donor's life but would also have prevented the whole transplantation-associated rabies "outbreak" in Germany.

Detection of virulence-associated genes characteristic of intestinal Escherichia coli pathotypes, including the enterohemorrhagic/enteroaggregative O104:H4, in bovines from Germany and Spain.

Cattle are reservoirs of enterohemorrhagic Escherichia coli; however, their role in the epidemiology of other pathogenic E. coli remains undefined. A new set of quantitative real-time PCR assays for the direct detection and quantification of nine virulence-associated genes (VAGs) characteristic of the most important human E. coli pathotypes and four serotype-related genes (wzxO104 , fliCH4 , rbfO157 , fliCH7 ) that can be used as a surveillance tool for detection of pathogenic strains was developed. A total of 970 cattle fecal samples were collected in slaughterhouses in Germany and Spain, pooled into 134 samples and analyzed with this tool. stx1, eae and invA were more prevalent in Spanish samples whereas bfpA, stx2, ehxA, elt, est and the rbfO157 /fliCH7 combination were observed in similar proportions in both countries. Genes characteristic of the hybrid O104:H4 strain of the 2011 German outbreak (stx2/aggR/wzxO104 /fliCH4 ) were simultaneously detected in six fecal pools from one German abattoir located near the outbreak epicenter. Although no isolate harboring the full stx2/aggR/wzxO104 /fliCH4 combination was cultured, sequencing of the aggR positive PCR products revealed 100% homology to the aggR from the outbreak strain. Concomitant detection by this direct approach of VAGs from a novel human pathogenic E. coli strain in cattle samples implies that the E. coli gene pool in these animals can be implicated in de novo formation of such highly-virulent strains. The application of this set of qPCRs in surveillance studies could be an efficient early-warning tool for the emergence of zoonotic E. coli in livestock.

Development of a rapid microarray-based DNA subtyping assay for the alleles of Shiga toxins 1 and 2 of Escherichia coli.

In this study, we developed a new rapid, economic, and automated microarray-based genotyping test for the standardized subtyping of Shiga toxins 1 and 2 of Escherichia coli. The microarrays from Alere Technologies can be used in two different formats, the ArrayTube and the ArrayStrip (which enables high-throughput testing in a 96-well format). One microarray chip harbors all the gene sequences necessary to distinguish between all Stx subtypes, facilitating the identification of single and multiple subtypes within a single isolate in one experiment. Specific software was developed to automatically analyze all data obtained from the microarray. The assay was validated with 21 Shiga toxin-producing E. coli (STEC) reference strains that were previously tested by the complete set of conventional subtyping PCRs. The microarray results showed 100% concordance with the PCR results. Essentially identical results were detected when the standard DNA extraction method was replaced by a time-saving heat lysis protocol. For further validation of the microarray, we identified the Stx subtypes or combinations of the subtypes in 446 STEC field isolates of human and animal origin. In summary, this oligonucleotide array represents an excellent diagnostic tool that provides some advantages over standard PCR-based subtyping. The number of the spotted probes on the microarrays can be increased by additional probes, such as for novel alleles, species markers, or resistance genes, should the need arise.

Occurrence of Enterobacteriaceae in Raw Meat and in Human Samples from Egyptian Retail Sellers.

The present study was performed to assess the presence of Enterobacteriaceae in raw meat and handlers in Egypt using cultivation and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). A total of 100 raw meat samples (chicken and beef meat, 50 each) were randomly purchased from butchers and local meat retailers located at Mansoura city, Egypt. Fifty human samples were collected from meat handlers (hand swabs and stool specimens, 25 each). 228 bacterial isolates were recovered from these samples. Unidentified isolates were characterized by partial 16S rRNA gene sequencing. Escherichia coli isolates were further typed using a DNA microarray system. Proteus spp. (60.0%) were found to be the most abundant followed by Escherichia coli (38.7%), Klebsiella spp. (17.3%), and Citrobacter spp. (13.3%). The presence of different Enterobacteriaceae in locally produced retail raw meat demonstrates the risk of infection of people through consumption of raw or undercooked meat and the risk for cross-contamination of other food products. Harmonized and concerted actions from veterinary and public health authorities are needed to reduce the risk of infection.

Rapid microarray-based DNA genoserotyping of Escherichia coli.

In this study, an improvement in the oligonucleotide-based DNA microarray for the genoserotyping of Escherichia coli is presented. Primer and probes for additional 70 O antigen groups were developed. The microarray was transferred to a new platform, the ArrayStrip format, which allows high through-put tests in 96-well formats and fully automated microarray analysis. Thus, starting from a single colony, it is possible to determine within a few hours and a single experiment, 94 of the over 180 known O antigen groups as well as 47 of the 53 different H antigens. The microarray was initially validated with a set of defined reference strains that had previously been serotyped by conventional agglutination in various reference centers. For further validation of the microarray, 180 clinical E. coli isolates of human origin (from urine samples, blood cultures, bronchial secretions, and wound swabs) and 53 E. coli isolates from cattle, pigs, and poultry were used. A high degree of concordance between the results of classical antibody-based serotyping and DNA-based genoserotyping was demonstrated during validation of the new 70 O antigen groups as well as for the field strains of human and animal origin. Therefore, this oligonucleotide array is a diagnostic tool that is user-friendly and more efficient than classical serotyping by agglutination. Furthermore, the tests can be performed in almost every routine lab and are easily expanded and standardized.

Zoonotic agents in small ruminants kept on city farms in southern Germany.

Sheep and goats are popular examples of livestock kept on city farms. In these settings, close contacts between humans and animals frequently occur. Although it is widely accepted that small ruminants can carry numerous zoonotic agents, it is unknown which of these agents actually occur in sheep and goats on city farms in Germany. We sampled feces and nasal liquid of 48 animals (28 goats, 20 sheep) distributed in 7 city farms and on one activity playground in southern Germany. We found that 100% of the sampled sheep and 89.3% of the goats carried Shiga toxin-producing Escherichia coli (STEC). The presence of Staphylococcus spp. in 75% of both sheep and goats could be demonstrated. Campylobacter spp. were detected in 25% and 14.3% of the sheep and goats, respectively. Neither Salmonella spp. nor Coxiella burnetii was found. On the basis of these data, we propose a reasonable hygiene scheme to prevent transmission of zoonotic agents during city farm visits.

No evidence of the Shiga toxin-producing E. coli O104:H4 outbreak strain or enteroaggregative E. coli (EAEC) found in cattle faeces in northern Germany, the hotspot of the 2011 HUS outbreak area.

BACKGROUND: Ruminants, in particular bovines, are the primary reservoir of Shiga toxin-producing E. coli (STEC), but whole genome analyses of the current German ESBL-producing O104:H4 outbreak strain of sequence type (ST) 678 showed this strain to be highly similar to enteroaggregative E. coli (EAEC). Strains of the EAEC pathotype are basically adapted to the human host. To clarify whether in contrast to this paradigm, the O104:H4 outbreak strain and/or EAEC may also be able to colonize ruminants, we screened a total of 2.000 colonies from faecal samples of 100 cattle from 34 different farms - all located in the HUS outbreak region of Northern Germany - for genes associated with the O104:H4 HUS outbreak strain (stx2, terD, rfbO104, fliCH4), STEC (stx1, stx2, escV), EAEC (pAA, aggR, astA), and ESBL-production (blaCTX-M, blaTEM, blaSHV). RESULTS: The faecal samples contained neither the HUS outbreak strain nor any EAEC. As the current outbreak strain belongs to ST678 and displays an en-teroaggregative and ESBL-producing phenotype, we additionally screened selected strains for ST678 as well as the aggregative adhesion pattern in HEp-2 cells. However, we were unable to find any strains belonging to ST678 or showing an aggregative adhesion pattern. A high percentage of animals (28%) shed STEC, corroborating previous knowl-edge and thereby proving the validity of our study. One of the STEC also harboured the LEE pathogenicity island. In addition, eleven animals shed ESBL-producing E. coli. CONCLUSIONS: While we are aware of the limitations of our survey, our data support the theory, that, in contrast to other Shiga-toxin producing E. coli, cattle are not the reservoir for the O104:H4 outbreak strain or other EAEC, but that the outbreak strain seems to be adapted to humans or might have yet another reservoir, raising new questions about the epidemiology of STEC O104:H4.

Determination of serotypes of Shiga toxin-producing Escherichia coli isolates by intact cell matrix-assisted laser desorption ionization-time of flight mass spectrometry.

Shiga toxin-producing Escherichia coli (STEC) isolates representing the serotypes O165:H25, O26:H11/H32, and O156:H25 were analyzed by matrix-assisted laser desorption/ionization (MALDI) mass spectra of whole cells, a procedure also known as intact cell mass spectrometry (ICMS or IC-MALDI MS) or MALDI-typing. We demonstrate that within the given species the three serotypes can be well discriminated by ICMS. Conditions for the construction of serotype-specific prototypic mass spectra were systematically optimized by filtering out masses that do not contribute to the discrimination of the serotypes. Binary distances between prototypic spectra and sample spectra were used to determine serotypes of unknown samples. With parameters optimized, only 0.7% of the assignments were incorrect compared to 31% when distances were calculated from alignments of unfiltered mass spectra. Within the different serotypes, clusters of genetically related E. coli most probably originating from single clones could be distinguished by restriction fragment length polymorphism analysis. Since ICMS did not reproduce these clusters, we conclude that the power of ICMS is just sufficient to discriminate E. coli serotypes under certain conditions but fails for the differentiation of E. coli below this level.

Rapid microarray-based genotyping of enterohemorrhagic Escherichia coli serotype O156:H25/H-/Hnt isolates from cattle and clonal relationship analysis.

Since enterohemorrhagic Escherichia coli (EHEC) isolates of serogroup O156 have been obtained from human diarrhea patients and asymptomatic carriers, we studied cattle as a potential reservoir for these bacteria. E. coli isolates serotyped by agglutination as O156:H25/H-/Hnt strains (n = 32) were isolated from three cattle farms during a period of 21 months and characterized by rapid microarray-based genotyping. The serotyping by agglutination of the O156 isolates was not confirmed in some cases by the results of DNA-based serotyping as only 25 of the 32 isolates were conclusively identified as O156:H25. In the multilocus sequence typing (MLST) analysis, all EHEC O156:H25 isolates were characterized as sequence type 300 (ST300) and ST688, which differ by a single-nucleotide exchange in the purA gene. Oligonucleotide microarrays allow simultaneous detection of a wider range of EHEC-associated and other E. coli virulence markers than other methods. All O156:H25 isolates showed a wide spectrum of virulence factors typical for EHEC. The stx(1) genes combined with the EHEC hlyA (hlyA(EHEC)) gene, the eae gene of the zeta subtype, as well as numerous other virulence markers were present in all EHEC O156:H25 strains. The behavior of eight different cluster groups, including four that were EHEC O156:H25, was monitored in space and time. Variations in the O156 cluster groups were detected. The results of the cluster analysis suggest that some O156:H25 strains had the genetic potential for a long persistence in the host and on the farm, while other strains did not. As judged by their pattern of virulence markers, E. coli O156:H25 isolates of bovine origin may represent a considerable risk for human infection. Our results showed that the miniaturized E. coli oligonucleotide arrays are an excellent tool for the rapid detection of a large number of virulence markers.

Analysis of the clonal relationship of serotype O26:H11 enterohemorrhagic Escherichia coli isolates from cattle.

Twelve cluster groups of Escherichia coli O26 isolates found in three cattle farms were monitored in space and time. Cluster analysis suggests that only some O26:H11 strains had the potential for long-term persistence in hosts and farms. As judged by their virulence markers, bovine enterohemorrhagic O26:H11 isolates may represent a considerable risk for human infection.

Genetic stability (in vivo) of the attenuated oral rabies virus vaccine SAD B19.

The distribution of oral rabies vaccine baits containing replication-competent live viruses poses certain environmental safety risks; among others, the possibility of reversion to or an increase in virulence. Hence, the genetic stability of the complete genome of the most widely used oral rabies vaccine virus, SAD B19, was examined after four and 10 serial i.c. passages in foxes and mice, respectively. It was shown that the consensus strain of SAD B19 was extremely stable in vivo. After 10 consecutive passages in mice not a single mutation was observed. In foxes, seven single nucleotide exchanges were found between the first and fourth passage, of which only one resulted in an amino acid exchange at position 9240 of the L-gene. This mutation was not observed during the first three passages and, furthermore, it was shown that this mutation was not linked to enhanced virulence.

Evaluation of five ELISA test kits for the measurement of antibodies against Mycobacterium avium subspecies paratuberculosis in bovine serum.

Five commercially available ELISA tests for the detection of antibodies against Mycobacterium avium subsp. paratuberculosis in bovine serum were evaluated at the individual animal level using sera from 286 paratuberculosis-free and 110 paratuberculosis-infected dairy cattle. Sensitivity (Se) and specificity (Sp) of the tests were estimated after determination of the cut-off dtheta by TG-ROC analysis or using the cut-off values recommended by the manufacturers, respectively. When the dtheta cut-off values were applied, the five ELISA tests showed sub-optimal Se and Sp. Adopting the cut-offs recommended by the manufacturers, the Sp of four of the five ELISA increased, two tests reaching Sp > or = 99.0%. Test sensitivity clearly depended on the disease state of the animals examined. Se was significantly higher in clinically diseased than in latently infected dairy cattle. Calculation of the positive and negative predictive values indicated that, depending on the test, a considerable proportion of false positive and false negative results have to be expected. Therefore, the suitability of antibody detection for the diagnosis of paratuberculosis in individual animals is questioned.

Genetic characterisation of attenuated SAD rabies virus strains used for oral vaccination of wildlife.

The elimination of rabies from the red fox (Vulpes vulpes) in Western Europe has been achieved by the oral rabies vaccination (ORV) of wildlife with a range of attenuated rabies virus strains. With the exception of the vaccinia rabies glycoprotein recombinant vaccine (VRG), all strains were originally derived from a common ancestor; the Street Alabama Dufferin (SAD) field strain. However, after more than 30 years of ORV it is still not possible to distinguish these vaccine strains and there is little information on the genetic basis for their attenuation. We therefore sequenced and compared the full-length genome of five commercially available SAD vaccine viruses (SAD B19, SAD P5/88, SAG2, SAD VA1 and SAD Bern) and four other SAD strains (the original SAD Bern, SAD VA1, ERA and SAD 1-3670 Wistar). Nucleotide sequencing allowed identifying each vaccine strain unambiguously. Phylogenetic analysis revealed that the majority of the currently used commercial attenuated rabies virus vaccines appear to be derived from SAD B19 rather than from SAD Bern. One commercially available vaccine virus did not contain the SAD strain mentioned in the product information of the producer. Two SAD vaccine strains appeared to consist of mixed genomic sequences. Furthermore, in-del events targeting A-rich sequences (in positive strand) within the 3' non-coding regions of M and G genes were observed in SAD-derivates developed in Europe. Our data also supports the idea of a possible recombination that had occurred during the derivation of the European branch of SAD viruses. If confirmed, this recombination event would be the first one reported among RABV vaccine strains.