Improvement of diagnostic tools for molecular epidemiology of listeriosis: Virulence genes and their implication in the pathogenesis of rhombencephalitis in ruminants.

• Unité de recherche: OSAV
• Numéro de projet: 1.08.11
• Titre du projet anglais: Improvement of diagnostic tools for molecular epidemiology of listeriosis: Virulence genes and their implication in the pathogenesis of rhombencephalitis in ruminants.

Mots-clé (Anglais)

Diagnosis and subtyping of L. monocytogenes, ruminants, encephalitis, virulence genes, molecular epidemiology, veterinary public health, zoonosis, reportable disease.

Description succincte (Anglais)

Listeriosis is an important food-borne infection in humans and animals and is reportable in Switzerland (art. 5 und 291, Schweizerische Tierseuchenverordnung). In humans the disease causes the highest hospitalisation and mortality rates amongst known food-borne pathogens, and in some European countries including Switzerland its incidence apparently rises. During a one-year active surveillance program for TSE’s in small ruminants, the number of diagnosed encephalitic listeriosis cases exceeded considerably the number of officially reported cases indicating that the prevalence of disease in small ruminants has been underestimated in Switzerland. Due to the ubiquity of L. monocytogenes, it is crucial to discriminate potentially pathogenic from apathogenic strains. Therefore, we as a reference laboratory for animal Listeriosis believe that the collection of reliable data on prevalence and distribution of L. monocytogenes strains in encephalitic listerosis in Switzerland is important in order to discover the role of ruminants as a reservoir of this important zoonotic pathogen and to apply accurate measures for control of listeriosis in animals and in humans, a central (Veterinary) Public Health issue. Both depend on reliability of the methods used for detection and precise identification of the bacteria and their subtypes. The aims of our project are therefore to 1) to establish a sensitive and rapid method for detection of L. monocytogenes in clinical cases, feedstuff (e.g. silage) and environmental samples based on real-time PCR, 2) to reliably subtype L. monocytogenes strains for outbreak investigation and prevention, and 3) to distinguish possibly pathogenic from apathogenic L. monocytogenes strains. To achieve the latter two objectives we propose the creation of a novel L. monocytogenes diagnostic tool, by combination of Multi-Locus Variable number of tandem repeats Analysis (MLVA) analysis with the analysis of virulence genes.

Objectifs du projet (Anglais)

Control of listeriosis in animals and in humans depends on reliability of the methods used for detection and precise identification of the bacteria and their subtypes. Our aims are 1) to establish a sensitive and rapid method for detection of L. monocytogenes applicable on clinical material, feedstuff (e.g. silage) and environmental samples (real-time PCR), 2) to reliably subtype L. monocytogenes strains for outbreak investigation and prevention, and 3) to distinguish possibly pathogenic from apathogenic L. monocytogenes strains. One central question of the project is whether there exist virulence differences amongst L. monocytogenes strains in ruminants (as it has been shown for humans), particularly with regard to the common encephalitis. Therefore the objects of our project are:

• Establishment of a quantitative real-time PCR for direct detection and quantification of Listeria monocytogenes (L. monocytogenes) without the need of culture
• The method will be established on different types of samples (formalin fixed and paraffin embedded tissues, brain, placenta, human and animal food-stuffs, e.g. silage)
• Establishment of Multi-Locus Variable number of tandem repeats Analysis (MLVA) for Listeria monocytogenes (L. monocytogenes) subtyping using additionally the mentioned relevant virulence genes as targets.
• Comparison to serotyping (Collaboration with the National Reference Centre for Listeriosis (CNRL); Prof. J. Bille) and biochemical strain differentiation.
• Analysis of the molecular epidemiology of listeriosis by screening of the most relevant virulence genes (in terms of prevalence and pathogenicity) of L. monocytogenes strains isolated in Switzerland. A central goal is the identification of virulence genes that play a role in the development of encephalitis, the most frequent form of listeriosis in ruminants.
• Comparison of isolated strains from different origin and pathologies (ruminant isolates from different Swiss regions; human and ruminant rhombencephalitis isolates versus environment isolates and isolates from other pathologies) by MLVA in combination with screening for virulence genes and cytopathogenicity assay is necessary to identify these genes.
• Correlation of virulence of the isolated strains with the lesion profile of encephalitis in affected ruminants.

Mise en oeuvre et application (Allemand)

31.05.2007: Umsetzung:
neue diagnostische Methode
Grundlagen für epidemiologische Abklärungen (Selektionssitzung 2007/mvo)

Mise en oeuvre et application (Anglais)

Listeriosis is an important food-borne disease in humans and ruminants due to its high mortality rate. Its control depends on reliability of the methods and the basic approaches used for detection and precise identifica-tion of the bacteria and their subtypes. The establishment of a quantitative PCR according to the European FOOD-PCR I project, ensures a rapid and standardized diagnosis of L. monocytogenes and lowers the de-tection levels of L. monocytogenes due to its increased sensitivity compared to the methods currently used in our laboratory. However, due to the ubiquity of L. monocytogenes, it is crucial to discriminate potentially pathogenic from apathogenic strains in order to apply adequate measures for disease control and prevention in ruminants and humans, a major (Veterinary) Public Health issue. The identification of such virulence fac-tors will permit the implementation of targeted surveys in order to investigate the prevalence of pathogenic L. monocytogenes strains in ruminants and the molecular epidemiology of listeriosis. Therefore we will screen for virulence genes in order to identify those that may play a role in the development of clinical disease, par-ticularly with regard to the pathogenesis of encephalitis in ruminants. The high prevalence of encephalitic listeriosis in small ruminants in Switzerland raises the question whether specific strains are associated with CNS infection. Furthermore, it is important to investigate whether the same strains cause encephalitis in ru-minants and humans, since the lesions of rhombencephalitis in both species are very similar and ruminants are believed to be a possible reservoir for pathogenic L. monocytogenes strains. The proposed MLVA virulence factor technique enables us to realize our commitment as reference laboratory for animal listeriosis to guaranty disease surveillance including: 1) the differentiation of potentially pathogenic from non-pathogenic L. monocytogenes, 2) clustering of isolates that are epidemiologically related, 3) identification of L. monocyto-genes isolates with an epidemiological relevancy, and 4) tracking of the source of L. monocytogenes infection. Its advantages over other techniques include its easiness, speed and reliability associated with relatively low costs, possible use in high throughput screenings and the facilitated comparison of results with other EU reference laboratories.

Publications / Résultats (Anglais)

Balandyte, L. (2010) MLVA typing of Listeria monocytogenes strains isolated from brain samples of sheep, goat and cattle, and comparison with strains of human, food and environmental origin. Inaugural-Dissertation, Vetsuisse-Fakultät, Universität Bern.   

Fauser, A. (2009) Evaluation of a multiplex real-time PCR for detection of Listeria monocytogenes and Listeria innocua in clinical brain tissue samples. Inaugural-Dissertation, Vetsuisse-Fakultät, Universität Bern.

Documents annexés

• Datenblatt [PDF] 15 kB
• Balandyte_Diss_Final.pdf [PDF] 2'979 kB
• Fauser_Diss_Final.pdf [PDF] 449 kB