Verotoxinogenic Escherichia coli (VTEC) O157:H7 – A Nationwide Swedish Survey of Bovine Faeces

In the autumn of 1995 the first outbreaks of enterohemorrhagic Escherichia coli O157:H7 including ca 100 human cases were reported in Sweden. From outbreaks in other countries it is known that cattle may carry these bacteria and in many cases is the source of infection. Therefore, the present study was performed to survey the Swedish bovine population for the presence of verotoxin-producing E. coli (VTEC) of serotype O157:H7. Individual faecal samples were collected at the 16 main Swedish abattoirs from April 1996 to August 1997. Of 3071 faecal samples, VTEC O157 were found in 37 samples indicating a prevalence of 1.2% (CI(95% )0.8–1.6). All 37 isolates carried genes encoding for verotoxin (VT1 and/or VT2), intimin, EHEC-haemolysin and flagellin H7 as determined by PCR. Another 3 strains were of serotype O157:H7 but did not produce verotoxins. The 37 VTEC O157:H7 strains were further characterised by phage typing and pulsed-field gel electrophoresis. The results clearly show that VTEC O157:H7 is established in the Swedish bovine population and indicate that the prevalence of cattle carrying VTEC O157:H7 is correlated to the overall geographical distribution of cattle in Sweden. Results of this study have formed the basis for specific measures recommended to Swedish cattle farmers, and furthermore, a permanent monitoring programme was launched for VTEC O157:H7 in Swedish cattle at slaughter

S. derby infection in Swedish pig herds

Within the Swedish salmonella control program any findings of salmonella is compulsory notifiable. In all Salmonella infected herds an official veterinary officer will perform an investigation and supervise the clearance of Salmonella infection/contamination from the farm (Wierup, M. 1991). Infected farms are subjected to restrictions including prohibition of movement of live animals except for transport to sanitary slaughter

A European interlaboratory trial to evaluate the performance of different PCR methods for Mycoplasma bovis diagnosis

Background Several species-specific PCR assays, based on a variety of target genes are currently used in the diagnosis of Mycoplasma bovis infections in cattle herds with respiratory diseases and/or mastitis. With this diversity of methods, and the development of new methods and formats, regular performance comparisons are required to ascertain diagnostic quality. The present study compares PCR methods that are currently used in six national veterinary institutes across Europe. Three different sample panels were compiled and analysed to assess the analytical specificity, analytical sensitivity and comparability of the different PCR methods. The results were also compared, when appropriate, to those obtained through isolation by culture. The sensitivity and comparability panels were composed of samples from bronchoalveolar fluids of veal calves, artificially contaminated or naturally infected, and hence the comparison of the different methods included the whole workflow from DNA extraction to PCR analysis. Results The participating laboratories used i) five different DNA extraction methods, ii) seven different real-time and/or end-point PCRs targeting four different genes and iii) six different real-time PCR platforms. Only one commercial kit was assessed; all other PCR assays were in-house tests adapted from published methods. The analytical specificity of the different PCR methods was comparable except for one laboratory where Mycoplasma agalactiae was tested positive. Frequently, weak-positive results with Ct values between 37 and 40 were obtained for non-target Mycoplasma strains. The limit of detection (LOD) varied from 10 to 103 CFU/ml to 103 and 106 CFU/ml for the real-time and end-point assays, respectively. Cultures were also shown to detect concentrations down to 102 CFU/ml. Although Ct values showed considerable variation with naturally infected samples, both between laboratories and tests, the final result interpretation of the samples (positive versus negative) was essentially the same between the different laboratories. Conclusion With a few exceptions, all methods used routinely in the participating laboratories showed comparable performance, which assures the quality of diagnosis, despite the multiplicity of the methods

Development and comparison of a real-time PCR assay for detection of Dichelobacter nodosus with culturing and conventional PCR: harmonisation between three laboratories

<p>Abstract</p> <p>Background</p> <p>Ovine footrot is a contagious disease with worldwide occurrence in sheep. The main causative agent is the fastidious bacterium <it>Dichelobacter nodosus</it>. In Scandinavia, footrot was first diagnosed in Sweden in 2004 and later also in Norway and Denmark. Clinical examination of sheep feet is fundamental to diagnosis of footrot, but <it>D. nodosu</it>s should also be detected to confirm the diagnosis. PCR-based detection using conventional PCR has been used at our institutes, but the method was laborious and there was a need for a faster, easier-to-interpret method. The aim of this study was to develop a TaqMan-based real-time PCR assay for detection of <it>D. nodosus </it>and to compare its performance with culturing and conventional PCR.</p> <p>Methods</p> <p>A <it>D. nodosus-</it>specific TaqMan based real-time PCR assay targeting the 16S rRNA gene was designed. The inclusivity and exclusivity (specificity) of the assay was tested using 55 bacterial and two fungal strains. To evaluate the sensitivity and harmonisation of results between different laboratories, aliquots of a single DNA preparation were analysed at three Scandinavian laboratories. The developed real-time PCR assay was compared to culturing by analysing 126 samples, and to a conventional PCR method by analysing 224 samples. A selection of PCR-products was cloned and sequenced in order to verify that they had been identified correctly.</p> <p>Results</p> <p>The developed assay had a detection limit of 3.9 fg of <it>D. nodosus </it>genomic DNA. This result was obtained at all three laboratories and corresponds to approximately three copies of the <it>D. nodosus </it>genome per reaction. The assay showed 100% inclusivity and 100% exclusivity for the strains tested. The real-time PCR assay found 54.8% more positive samples than by culturing and 8% more than conventional PCR.</p> <p>Conclusions</p> <p>The developed real-time PCR assay has good specificity and sensitivity for detection of <it>D. nodosus</it>, and the results are easy to interpret. The method is less time-consuming than either culturing or conventional PCR.</p

Experimental infection in calves with a specific subtype of verocytotoxin-producing Escherichia coli O157:H7 of bovine origin

<p>Abstract</p> <p>Background</p> <p>In Sweden, a particular subtype of verocytotoxin-producing <it>Escherichia coli </it>(VTEC) O157:H7, originally defined as being of phage type 4, and carrying two <it>vtx</it>₂genes, has been found to cause the majority of reported human infections during the past 15 years, including both sporadic cases and outbreaks. One plausible explanation for this could be that this particular subtype is better adapted to colonise cattle, and thereby may be excreted in greater concentrations and for longer periods than other VTEC O157:H7 subtypes.</p> <p>Methods</p> <p>In an experimental study, 4 calves were inoculated with 10⁹colony forming units (cfu) of strain CCUG 53931, representative of the subtype VTEC O157:H7 (PT4;<it>vtx</it>₂;<it>vtx</it>_2c). Two un-inoculated calves were co-housed with the inoculated calves. Initially, the VTEC O157:H7 strain had been isolated from a dairy herd with naturally occurring infection and the farm had previously also been linked to human infection with the same strain. Faecal samples were collected over up to a 2-month period and analysed for VTEC O157 by immuno-magnetic separation (IMS), and IMS positive samples were further analysed by direct plating to elucidate the shedding pattern. Samples were also collected from the pharynx.</p> <p>Results</p> <p>All inoculated calves proved culture-positive in faeces within 24 hours after inoculation and the un-inoculated calves similarly on days 1 and 3 post-inoculation. One calf was persistently culture-positive for 43 days; in the remainder, the VTEC O157:H7 count in faeces decreased over the first 2 weeks. All pharyngeal samples were culture-negative for VTEC O157:H7.</p> <p>Conclusion</p> <p>This study contributes with information concerning the dynamics of a specific subtype of VTEC O157:H7 colonisation in dairy calves. This subtype, VTEC O157:H7 (PT4;<it>vtx</it>_2;<it>vtx</it>_2c), is frequently isolated from Swedish cattle and has also been found to cause the majority of reported human infections in Sweden during the past 15 years. In most calves, inoculated with a representative strain of this specific subtype, the numbers of shed bacteria declined over the first two weeks. One calf could possibly be classified as a high-shedder, excreting high levels of the bacterium for a prolonged period.</p

S. derby infection in Swedish pig herds

Within the Swedish salmonella control program any findings of salmonella is compulsory notifiable. In all Salmonella infected herds an official veterinary officer will perform an investigation and supervise the clearance of Salmonella infection/contamination from the farm (Wierup, M. 1991). Infected farms are subjected to restrictions including prohibition of movement of live animals except for transport to sanitary slaughter.</p

Differences in the genome, methylome, and transcriptome do not differentiate isolates of Streptococcus equi subsp. equi from horses with acute clinical signs from isolates of inapparent carriers

Streptococcus equi subsp. equi (SEE) is a host-restricted bacterium that causes the common infectious upper respiratory disease known as strangles in horses. Perpetuation of SEE infection appears attributable to inapparent carrier horses because it neither persists long-term in the environment nor infects other host mammals or vectors, and infection results in short-lived immunity. Whether pathogen factors enable SEE to remain in horses without causing clinical signs remains poorly understood. Thus, our objective was to use next-generation sequencing technologies to characterize the genome, methylome, and transcriptome of isolates of SEE from horses with acute clinical strangles and inapparent carrier horses-including isolates recovered from individual horses sampled repeatedly-to assess pathogen-associated changes that might reflect specific adaptions of SEE to the host that contribute to inapparent carriage. The accessory genome elements and methylome of SEE isolates from Sweden and Pennsylvania revealed no significant or consistent differences between acute clinical and inapparent carrier isolates of SEE. RNA sequencing of SEE isolates from Pennsylvania demonstrated no genes that were differentially expressed between acute clinical and inapparent carrier isolates of SEE. The absence of specific, consistent changes in the accessory genomes, methylomes, and transcriptomes of acute clinical and inapparent carrier isolates of SEE indicates that adaptations of SEE to the host are unlikely to explain the carrier state of SEE. Efforts to understand the carrier state of SEE should instead focus on host factors