15/06/2022

Bacterial Zoonoses Unit (UZB)

Head of Unit: Claire Ponsart

The unit deals with bacterial infections that can be transmitted from animals to humans and constitute a major public health risk, such as brucellosis, chlamydia, anthrax, melioidosis, glanders, bovine tuberculosis and tularaemia. These animal diseases can cause severe, sometimes fatal, human cases, requiring major and/or prolonged treatment, and can result in occupational incapacity of varying durations or even severe disabilities. Animal cases can lead to significant economic losses through mortality, induced abortions or sterility, and through culling and/or trade restrictions imposed by national, European or international regulations. All the diseases studied (except bovine tuberculosis and chlamydia) are caused by bacteria that can potentially be used for malicious purposes (bioterrorism) and fall under the regulations for "highly pathogenic micro-organisms and toxins" (MOT).

Reference activities

The unit has national reference laboratory mandates for six diseases (brucellosis, anthrax, avian chlamydia, glanders/melioidosis, bovine tuberculosis and tularaemia). It is the European Union Reference Laboratory (EURL) for brucellosis and equine diseases (glanders). Lastly, it has six WOAH reference mandates (brucellosis, contagious epididymitis in rams, avian chlamydiosis, chlamydial abortion, glanders/melioidosis and bovine tuberculosis).

Surveillance activities

The unit is involved in various surveillance programmes of the Epidemiological surveillance platform for animal health (ESA Platform) on the themes it covers, mainly brucellosis and tuberculosis. In particular, it contributes to the surveillance programme for bovine tuberculosis in wildlife (Sylvatub) and the SAGIR "Surveillance for action" network for brucellosis and tularaemia.

Expert appraisal activities

The unit contributes to several groups of the ESA Platform (Tuberculosis, Sylvatub, Observatory on the causes of abortion, etc.). The unit's scientists also participate in several expert groups, in particular on anthrax, brucellosis and tuberculosis, making a significant contribution to the surveillance mission and to the revision of control programmes for these diseases.

Research activities

The unit's research is directly tied to the issues raised by the results of its reference work, and concerns animal health and welfare, epidemiological surveillance, antimicrobial resistance and food safety. These areas are covered both through its reference activities (scientific and technical support for the zoonotic bacteria covered by the unit, expert appraisals on surveillance programmes for brucellosis and tuberculosis) and as part of epidemiological and phylogeographical studies carried out on various topics.

The themes studied by the UZB include:

  • characterisation of Brucella sp., Chlamydia sp., Mycobacterium sp., Bacillus anthracis and vector-borne zoonotic bacteria: Francisella tularensis; Development of tools for phenotypic or molecular differentiation of bacterial strains to enable more precise epidemiological monitoring of outbreaks and the establishment of links between animal outbreaks and human cases;
  • development of "One Health" approaches for studying interactions between pathogens, host species and the environment. Several projects are dedicated to wildlife surveillance and the role of different species in pathogen transmission. Others focus on the presence and persistence of pathogens in the environment, particularly in water bodies, with the possibility of investigating pathogen survival in amoebae;
  • applications of new high-throughput technologies to reference activities (in particular molecular typing of strains; design of microarrays for multi-pathogen detection or differential diagnosis) and research (phylogeographical approaches; studies of virulence genes or genes involved in survival; modelling work associated with transmission pathways). Use of next generation sequencing (NGS), which enables large quantities of genomes to be sequenced in record time and at a more affordable price;
  • epidemiological studies in production livestock in order to validate diagnostic and/or screening tools, and in wildlife and/or vectors for health surveillance and for identifying reservoir populations and/or victims of bacterial infections, in partnership with other ANSES laboratories and other organisations.

Main research projects

Funding: DIM1Health field of major interest (2021-2023 Doctoral contract )

Partnership: French Institute for Research for Development (IRD), collaboration with the French Armed Forces Biomedical Research Institute (IRBA) and the Institut Pasteur of Guadeloupe.

In order to integrate the non-uniform distribution of B. pseudomallei and predict priority areas for surveillance and active diagnosis of the disease, it is essential to understand the spatial distribution of the pathogen in soil and water, identify its ecological niche and determine the influence of climatic and/or seasonal factors.

As part of this project, a retrospective study of human cases that have occurred in the French overseas territories will be carried out to target priority areas that will be included in the environmental investigation (analysis of water and soil samples). A serological investigation will be carried out on domestic animals in these areas, including small ruminants that are highly susceptible to the disease, as well as on wild animals. All the data collected (on humans, animals and the environment) will be aggregated to identify sources of exposure and risk factors, and to obtain tools to assist in monitoring and decision-making.

Funding: One Health European Joint Programme (OHEJP)

The Idembru project, an OHEJP project coordinated by our team, is focusing on the pathogen Brucella, which causes brucellosis. It should help complete the genomic characterisation of so-called "atypical" Brucella isolated from different forest, marine and wetland (riverbank) ecosystems. It also aims to understand the zoonotic potential and virulence of a panel of emerging and atypical Brucella strains, using in vivo and in vitro infection models. Lastly, it will develop a toolkit focusing on emerging Brucella species and reservoirs to ensure rapid detection, identification and characterisation.

Trans-Pyrenean network for research and development of innovative tools for the control of animal tuberculosis

Partners: IRTA (Spain), NEIKER (Spain), UAB (Spain), ENVT (France)

Funding: European Union through the Interreg Programme (POCTEFA 2014-2020)

The InnoTub project aims to create and consolidate a research, development and innovation network involving research centres, higher education institutions and technology companies in the Trans-Pyrenees region, in order to provide solutions for the agri-food sector and public health in combating animal tuberculosis. The project will also seek to develop tools and strategies to improve the management of risks of infection in cattle and increase the acceptability of the animal tuberculosis control programme, as well as to develop new marketable diagnostic tools, vaccines and innovative compounds for controlling tuberculosis in domestic and wild animals.

Partner: INRAE Nouzilly

Funding: One Health European Joint Programme – ANSES

Far from being a disease of the past, bovine tuberculosis (bTB) is a zoonosis whose economic impact on French agriculture remains especially high, even today. In recent years, increasingly common cases of bTB identified in both farm animals and wildlife have further complicated the problem of this notifiable disease and its health management. New questions are arising about its epidemiology and the ability of certain strains to proliferate in a multi-host system involving cattle, wildlife and their environment. We recently obtained the first genomic data on several dozen French strains and highlighted the presence of several major clonal complexes in the country, as well as the emergence of dominant genotypes.

The aim of the PeMBO project is to establish the phenotypic profile of these emerging genotypes of Mycobacterium bovis with a broad host range, using new genomic and biochemical approaches. This project consists of three tasks: the first is to obtain reference sequences by de novo assembly (Illumina/PacBio sequencing) of genomes of strains belonging to the majority clonal groups. The second is to search for genomic events (insertion/deletion or large sequence polymorphism (LSP)) on the whole genome, with a study of genetic variation focused on the analysis of virulence genes, genes involved in envelope biosynthesis and excreted antigens. Lastly, the third task is to analyse protein and lipidomic profiles.

Partners: NIBSC (UK), APHA (UK), Senasa (Argentina), EURL for Bovine Tuberculosis – Visavet (Spain), APHIS (USA).

Funding: World Organisation for Animal Health (WOAH)

The International Standard for Bovine Tuberculin (ISBT) was produced in 1986. It serves as a reference standard for quality control testing of the purified protein derivative (PPD) of bovine tuberculin used in bovine tuberculosis (bTB) surveillance, diagnosis and export certification. It is running out and needs to be replaced in the short term.

A project to replace the ISBT has been launched by the WOAH. This involves an ad hoc group of bTB experts including the NRL for Tuberculosis (ANSES), the WOAH Reference Laboratories for Bovine Tuberculosis (France, Argentina and the UK), the National Institute for Biological Standardisation and Control (NIBSC) for the preparation, storage and distribution of tuberculins; and collaborating scientists from about 15 other national laboratories.

Two candidate tuberculins will be tested against the current ISBT, in order to assess and calibrate their potency and specificity and determine their suitability for the field.

Partners: EPI Unit

Funding: French Animal Health Network (RFSA)

In recent years, numerous molecular techniques have been developed to differentiate isolates belonging to the Mycobacterium tuberculosis complex (MTBC). For M. bovis, the techniques used routinely are spoligotyping and multiple loci VNTR analysis (MLVA). The vast theoretical diversity of molecular profiles obtained by combining these two techniques makes it possible to determine the origin of infection of a huge number of outbreaks and identify possible interspecies transmission, by comparing the profiles of strains isolated from cattle and wildlife. In the field, however, the situation is different: in areas with the highest incidence of the disease, almost all isolates share the dominant genotype profiles in each area, making them largely irrelevant for reconstructing the transmission chain. It then becomes necessary to use very fine resolution molecular techniques to be able to trace the infection within a small geographical area. Whole genome sequencing of the bacterium seems to be the most suitable technique, as it allows the detection of genomic changes on a very small scale. The data generated can be used to reconstruct transmission scenarios for the infection.

The objectives of this project were to determine within the same geographical area the links (i) between infected cattle herds, and (ii) between infected cattle herds and infected wildlife, through an analysis of the complete genome sequences of M. bovis strains isolated in the area. This study sought to differentiate between a resurgence of infection and a new infection, and to provide insights into the role of wildlife in the circulation of the infection within the area.

Partners: INRAE Dijon

Funding: French Animal Health Network (RFSA)

Bovine tuberculosis testing is based on detection of the cell-mediated immune response using single (SITT) or comparative (CITT) intradermal tuberculin tests and the interferon gamma (IFN-γ) assay. Intradermal tuberculin tests have been found to be an adequate screening tool at the herd level, and several countries have successfully eradicated the disease on the basis of their use. Inclusion of the IFN-γ test in parallel has proven to be an adequate method for maximising the detection of infected cattle. However, a major limitation of these methods is their lack of specificity, which can lead to false positive results. Many so-called nontuberculous mycobacteria (NTMs) have been reported as agents that interfere with these methods. Common antigens with these NTMs have long been described as responsible for non-specific reactions, especially with SITT. Introduction of the CITT has improved this test's specificity by excluding reactions caused by M. avium complex mycobacteria.

The aim of this project was to develop a tool to identify the agents potentially responsible for these atypical reactions in environmental samples. The BioMark microfluidic system (Fluidigm) was used because of its ability to test a large number of samples against a large number of targets. The primary objective was to develop a high-throughput PCR tool. The second objective was to test samples taken from the cattle environment, targeting herds that have reacted to ante mortem testing.

Partners: ANSES's Nancy Laboratory for Rabies and Wildlife, APHA (UK), University of Surrey (UK)

Funding: French Animal Health Network (RFSA)

In addition to the project on the epidemiology of TB in foxes, this experimental model development project was set up to better understand the pathology of the disease in this species. The project's primary objective was to establish an experimental infection protocol (dose and type of virulent M. bovis strains, route of inoculation, infected organs and pathology), as well as to collect biological material (e.g. sera) to be characterised for diagnostic validation purposes. The second objective was to monitor bacterial shedding (oropharyngeal, faecal and urinary tract) throughout the duration of the infection. The third objective was to develop immunological tools to target markers of vaccination and protection for use in vaccine efficacy studies.

Partners: ANSES's Nancy Laboratory for Rabies and Wildlife, APHA (UK), University of Surrey (UK), AFBI (Northern Ireland), UCD (Ireland)

Funding: French Animal Health Network (RFSA) and VETBIONET

While the experimental infection model is central to confirming the protective effect of vaccines, the relative pathogenicity of the different models has never been compared in mustelids. Unification of the infection model would simplify the vaccine development process in the future. The ferret is a laboratory mustelid (easier to procure – farmed animal – and handle compared to wild-caught animals) and a natural host for M. bovis, and has already been used by the Northern Ireland team. Development of the tuberculosis infection model in ferrets will enable the experimental results already obtained in badgers to be standardised. The project's primary objective was to establish the experimental model (dose and strains of virulent M. bovis). The second objective was to develop immunological tools to target markers of vaccination and protection for use in vaccine efficacy studies for which the infection model is required.

Partners: EPI Unit, INRAE

Funding: ANSES

Over the past decade, new atypical Brucella species, including several strains resembling Brucella inopinata, have been detected in both wild-caught and "exotic" amphibians from various continents. In 2017, a strain of Brucella was isolated for the first time by our laboratory from animals on a farm producing frogs for human consumption. It was surprisingly characterised and identified as being similar to B. microti.

Partners: French Biodiversity Agency (OFB), French Natural History Museum (MNHN), National Research Institute for Agriculture, Food and the Environment (INRAE)

Funding: French Ministry of Agriculture and Fisheries; Ministry of Ecological and Inclusive Transition

Following a joint request by the Ministers for the Environment and Agriculture, in the spring of 2017 ANSES, the OFB, INRAE and the MNHN carried out testing of a vaccine against brucellosis in the Alpine ibex (Capra ibex). The goal was to provide new information for managing the brucellosis outbreak affecting this species in the Bargy Massif (Haute Savoie). In particular, the study sought to compare the safety of the Rev.1 conjunctival vaccine, already licensed for use in domestic sheep and goats, between the domestic goat (Capra hircus) and the Alpine ibex.

Partners: ANSES's Nancy Laboratory for Rabies and Wildlife, DRAAF Nouvelle Aquitaine, Dordogne departmental hunting federation, Dordogne departmental testing and research laboratory, National hunting federation, Dordogne department of the National office for hunting and wildlife, Dordogne departmental health protection group, Dordogne licensed trappers association, Inter-regional veterinary epidemiology unit (Côte d'Or)

Funding: French Animal Health Network (RFSA)

Although susceptible to M. bovis, the fox (Vulpes vulpes) has only rarely been found infected in areas of France endemic for bovine tuberculosis (1/160 in the Brotonne forest, 2/62 in the Côte d'Or, 0/64 in the Dordogne). However, in 2015 in Dordogne, four foxes (out of six tested) were found to be infected.

They came from a municipality in an area where cattle, badgers, wild boar and deer had also been found to be infected. The excreta (saliva, urine, faeces) of one of the four foxes were found positive for M. bovis by PCR. This discovery, and results obtained in Côte-d'Or on the frequency and behaviour of foxes visiting livestock buildings and feed troughs, raise the question of the epidemiological role of foxes in endemic areas. This project's general objective was to study the epidemiological role of the fox in tuberculosis circulation in several endemic areas, namely Dordogne, Charente, Pyrénées-Atlantiques, Landes and Côte d'Or. The specific objectives were to (1) estimate the prevalence of lesions and infections, (2) study routes of exposure and shedding, and (3) study the degree of association with epidemiological (infection in other species), individual (age) and population (density estimator) factors.