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Scientific Achievements Year 3

As we reach the end of 2007, we look back a few selected scientific achievements of Med-Vet-Net in Year 3 of the project.

PCR results from Wokpackage 29 illustrating presence of the transposon bearing armA gene.

A fully harmonized and standardized, real-time surveillance database system to detect disease clusters of Salmonella, VTEC and Listeria monocytogenes for food, public health and veterinary laboratories has been delivered to ECDC.

PulseNet Europe – Med-Vet-Net’s Workpackage 4 – developed a network of public health, veterinary and food laboratories for molecular surveillance of food-borne infections in Europe. This network has established a customized, web-based database system to detect disease clusters and investigate outbreaks of Salmonella, verocytoxin-producing E. coli and Listeria.

Now that this Workpackage is complete, all PulseNet Europe activities and molecular databases will be transferred to the European Centre for Disease Prevention and Control (ECDC) in Stockholm. This transfer is also supported by the European Food Safety Authority, which is important as PulseNet Europe has many partners from food and veterinary laboratories.

A web-based ‘atlas’ of the incidence of salmonellosis in Europe has been developed in collaboration with EnterNet.

Workpackage 6 aims to establish a geographical informations systems (GIS) network and build up GIS capacities for analysing exposure to major bacterial food-borne pathogens within various epidemiological contexts.

The first GIS project has now been completed An Internet-atlas of salmonellosis in Europe with a series of maps showing the incidence of laboratory-confirmed human cases by region and year for the 10 most common serotypes. The atlas is available at: www.epigis.dk

Standardized rapid methods for the detection and characterization of Salmonella Genomic Island 1 have been delivered.

Salmonella Genomic Island 1 is a gene cluster found in S. Typhimurium DT104, which is zoonotic in origin and causes numerous infections in humans. DT104 is associated with an enhanced ability to cause disease (virulence) and multi-drug resistance. Workpackage 21 has developed a phenotypic method to detect SGI1-positive isolates from strain collections, based on their resistance profile. A real-time PCR protocol for the sensitive and specific detection of SGI1 has also been optimized. And, a novel fingerprinting method to characterize the non-multi drug resistance region of SGI1 has been developed.
A paper on the results of the molecular analysis of a strain collection of 445 multi drug-resistant European isolates of Salmonella, Shigella and Proteus will be published in 2008.

Harmonized and standardized PCR-based methods for the detection of Cryptosporidium and Giardia have been established.

Zoop-Net – Workpackage 22 – aims to implement expertise on the detection and control of Cryptosporidium and Giardia at the European level.
A common molecular platform has now been established in Europe and comparable data on Cryptosporidium and Giardia can now be generated to investigate the epidemiology of these infections and to study the role of potential animal sources in human disease.
The dynamic database, containing DNA sequences and genotype data linked to epidemiological data, is available at: https://hypocrates.rivm.nl/bnwww/MedVetNet/

A strategic document ‘Methodological choices for calculating the disease burden and cost of illness of food-borne zoonoses in European countries’ has been adopted.

This document from Workpackage 23 – Prioritizing food-borne and zoonotic hazards at the EU level – indicates that disease burden and cost-of-illness calculations make several choices about the exact methodology. These choices have an impact on the final results and must be appropriate for the decision context of the study, and should reflect the values that exist in the societies under study.
The document is available at: www.medvetnet.org/pdf/Reports/Report_07-002/pdf

A comparison of the European Campylobacter risk assessment has been completed.

Workpackage 24 aims to compare existing national quantitative risk assessment models on Campylobacter inbroiler meat in Europe, and to develop a European consensus approach. For a comparison of models.
For a comparison of models, expert partners held three subgroup meetings covering: (1) the farm phase; (2) the processing and retail phase; and (3) the consumer preparation and illness phase. Models were discussed in detail to identify the important differences and similarities, and to find the strengths and weaknesses in the available risk assessments. Results have been presented and discussed in a meeting with risk managers and other interested parties. This has resulted in a report (www.medvetnet.org/pdf/Reports/D24-1.pdf) and a review paper on Campylobacter risk assessments in Europe has been prepared.

The virulence gene content of 1021 strains of E. coli and Salmonella from eight partners in seven countries has been investigated and analysed by standardized PCRs.

The identification of new and emerging pathotypes of Salmonella and verocytoxin producing E. coli (VTEC) is essential if intervention strategies are to be developed. In Workpackage 26, a number of virulence-associated genes have been selected for the screening of strains. Criteria for the PCR of these genes (virulotyping) including primers and conditions were distributed to partners along with control DNA. In total 1021 isolates have been selected and tested.
All the isolate and PCR data have been curated in a database and some exploratory analysis has been performed. The data have been re-validated to ensure there were no transcriptional errors in the database. The preliminary analysis indicates significant differences in the distribution of virulence genes in E. coli and Salmonella strains across Europe.

Significant progress has been in the detection and epidemiology of Trichinella using novel diagnostic reagents and tests as well as risk assessment approaches.

Workpackage 27– TrichiMED – looks at harmonization of Trichinella infection control methods, quantitative risk assessment in pigs and an early diagnosis in humans to increase treatment efficacy. The workpackage has developed serodiagnostic tests for the early detection of Trichinella infection, including methods for recombinant antigen expression.
TrichiMED also works on harmonizing veterinary control methods for Trichinella, particularly between National Reference Laboratories, in light of new EU regulations. Proficiency samples were distributed in two ring trials to compare the sensitivity of different official detection methods.
In addition, ISS, a partner in Workpackage 27, has developed and validated an indirect ELISA test based on the T. spiralis excretory/secretory antigens. This ELISA was evaluated by a ring trial involving 23 European laboratories. Results indicate that the test in reproducible and robust suggesting that it can be used in active surveillance.

Poland has been identified as potential European reservoir of the 16S rRNA methylase, armA, conferring resistance to aminoglycosides.

In order to determine the prevalence of isolates containing gene armA (one of a family of determinants for aminoglycoside resistance via 16S rRNA methylase), Workpackage 29 partners used their respective electronic databases to screen any organisms for suspect resistance phenotype patterns (as indicated by high-level aminoglycoside-resistance). In total, nearly one million strains from 12 countries were analysed in this way. Those isolates that fulfilled this criterion were then tested by PCR using a harmonized PCR-protocol. The results showed that none of the approximately 500 000 animal isolates stored in the database bore evidence for aminoglycoside-conferring 16SrRNA resistance. However, out of the approximately 500 000 human isolates investigated in the electronic database, subsequent genetic analysis of potentially positive isolates revealed that 27 carried the armA gene. Five of these strains, investigated at HPA, were strains originating from a hospital outbreak in Poland. The remaining 22 isolates were all identified at the PZH in Poland. These results indicate that, of the 12 EU countries tested, Poland is the only major reservoir to date of this antimicrobial resistance determinant.

Serological tests for zoonotic viruses such as HEV have been improved with the use of recombinant antigens.

Workpackage 31 aims to generate knowledge and methodologies on Hepatitis E virus (HEV), Anellovirus, and Encephalomyocarditis virus. There are currently no methods for the culture of HEV. However, the cloning and expression of antigens and capsid proteins, by molecular engineering techniques has been undertaken. Several cDNA clones from swine genotype 3 and human genotype 1 (Sar-55) viral strains have been generated by various partners in Workpackage 31 and work is in progress to express these proteins in large amounts and in antigenically conserved forms. In particular, using a baculovirus expression system, significant amounts of virus-like particles (VLP) of a French genotype 3 HEV have been synthesized by AFSSA. These VLP appear well preserved by electron microscopy, and react efficiently in both ELISA assays and western blotting with the sera from infected humans, swine and wild boar. Major production of a truncated form of G1 HEV has also been achieved at the VLA. These antigens, in either assembled or soluble forms, appear suitable for establishing sensitive and specific immunoassays for HEV antibody identification.

The assembled VLP preparation is being used, in collaboration with ISS, to produce monoclonal antibodies (MAbs) to conformation-dependent antigenic sites of the HEV capsid.

A novel approach has been delivered, using sero-epidemiology, to determine more accurately the public health burden of food-borne disease.

The main objective of Workpackage 32 is to provide estimates of the population-based incidence of Campylobacter and non-typhoid Salmonella infections in European countries, which are independent of the sensitivity and specificity of the respective countries’ surveillance systems, in order to allow a comparison of the population burden of infections across these countries. The methodological approach to achieve this is sero-epidemiology: from measured levels of specific antibodies in population-representative serum collections, estimates of the incidence of infection are generated with the help of a mathematical back-calculation model.

During the first year of the Workpackage, the methodology has been refined and finalized. The model development and its preliminary use to determine exposure to Salmonella using Danish sera has been published (Simonsen et al. 2007; Epidemiol. Infection; Online 3 Aug; http://tinyurl.com/3yoqgn). A protocol for a European pilot study involving sera from at least six EU countries has been agreed and suitable serum collections have been identified in Denmark, Netherlands, France, Finland, Poland, Romania and the USA (as an external partner).

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Page Contact: Jennie Drew - Last modified: 2007-12-31