The Rise of Three Rs Centres and Platforms in Europe.

Public awareness and discussion about animal experiments and replacement methods has greatly increased in recent years. The term 'the Three Rs', which stands for the Replacement, Reduction and Refinement of animal experiments, is inseparably linked in this context. A common goal within the Three Rs scientific community is to develop predictive non-animal models and to better integrate all available data from in vitro, in silico and omics technologies into regulatory decision-making processes regarding, for example, the toxicity of chemicals, drugs or food ingredients. In addition, it is a general concern to implement (human) non-animal methods in basic research. Toward these efforts, there has been an ever-increasing number of Three Rs centres and platforms established over recent years - not only to develop novel methods, but also to disseminate knowledge and help to implement the Three Rs principles in policies and education. The adoption of Directive 2010/63/EU on the protection of animals used for scientific purposes gave a strong impetus to the creation of Three Rs initiatives, in the form of centres and platforms. As the first of a series of papers, this article gives an overview of the European Three Rs centres and platforms, and their historical development. The subsequent articles, to be published over the course of ATLA's 50th Anniversary year, will summarise the current focus and tasks as well as the future and the plans of the Three Rs centres and platforms. The Three Rs centres and platforms are very important points of contact and play an immense role in their respective countries as 'on the ground' facilitators of Directive 2010/63/EU. They are also invaluable for the widespread dissemination of information and for promoting implementation of the Three Rs in general.

The Current Status and Work of Three Rs Centres and Platforms in Europe.

The adoption of Directive 2010/63/EU on the protection of animals used for scientific purposes has given a major push to the formation of Three Rs initiatives in the form of centres and platforms. These centres and platforms are dedicated to the so-called Three Rs, which are the Replacement, Reduction and Refinement of animal use in experiments. ATLA's 50th Anniversary year has seen the publication of two articles on European Three Rs centres and platforms. The first of these was about the progressive rise in their numbers and about their founding history; this second part focuses on their current status and activities. This article takes a closer look at their financial and organisational structures, describes their Three Rs focus and core activities (dissemination, education, implementation, scientific quality/translatability, ethics), and presents their areas of responsibility and projects in detail. This overview of the work and diverse structures of the Three Rs centres and platforms is not only intended to bring them closer to the reader, but also to provide role models and show examples of how such Three Rs centres and platforms could be made sustainable. The Three Rs centres and platforms are very important focal points and play an immense role as facilitators of Directive 2010/63/EU 'on the ground' in their respective countries. They are also invaluable for the wide dissemination of information and for promoting the implementation of the Three Rs in general.

Single nucleotide polymorphism (SNP) characterisation of mouse inbred strains bred at MRC-National Institute for Medical Research.

Inbred mouse strains have long proved useful as tools for biomedical research. They remove the effects of genetic background as an experimental variable. Within all mouse colonies, genetic drift is a recognised phenomenon and monitoring and documenting changes is important for experimental design and consistency. This communication documents the initial characterisation through SNP analysis of the inbred mouse strains bred and used at the time at the Medical Research Council National Institute for Medical Research (MRC-NIMR), Mill Hill, now The Crick Institute. These inbred strains were part of the foundation colonies for the many genetically modified mouse strains made at Mill Hill. We found small genetic changes in four of the nine inbred strains. Although phenotypic differences have not yet been found between the NIMR and the correspondent parental strains, I cannot discard that these may arise or have already arisen. This work has also authenticated the 129/SvJEvNimr-Gpi1c strain that was widely used at MRC-NIMR for gene targeting. All these inbred strains have been renamed according to The International Committee on Standardized Genetic Nomenclature for Mice.

The role of environmental enrichment in the aetiopathogenesis of Staphylococcus aureus facial, periorbital and retroorbital abscesses in mice.

In this observational retrospective study, an outbreak of Staphylococcus aureus abscesses was correlated with the presence of sharp edges in damaged plastic environmental enrichment within the cages. In 2010, Lawson reported cases of S. aureus mandibulofacial and maxillofacial abscess in mice and proposed excessive barbering or grooming, leading to the mastication and fragmentation of hair, as an aetiopathogenesis of S. aureus abscesses. In contrast, in this study, the presence of hair was not found in any of the histopathology, and abscesses were present in the periorbital area. S. aureus colonises the skin, nasopharynx and intestines, and may cause pyogenic infections if a breach in local defences promotes staphylococcal invasion. Whole genome sequencing and analysis supported the hypothesis that this outbreak resulted from clonal expansion of S. aureus infected C57BL6/J mice imported into the area and infection transmission from humans to mice was ruled out. An additional aetiopathogenesis is proposed for S. aureus abscesses with the sharp edges of damaged plastic environmental enrichment items leading to oral mucosal injury allowing S. aureus entrance into tissues, its carriage into the submucosa, followed by abscess formation.

Quantification of Hair Corticosterone, DHEA and Testosterone as a Potential Tool for Welfare Assessment in Male Laboratory Mice.

Steroids, providing information regarding several biological patterns including stress and sexual behavior, have been investigated in different matrices in laboratory mice. Data regarding hair quantification, indicative of longer timespans when compared to blood and saliva, are lacking. The aim of the work was to analyze the hormonal hair profile of laboratory male mice and to investigate potential relationships with age and housing, as a potential tool for welfare assessment. Fifty-six adult male C57BL/6J and C57BL/6OlaHsd substrain mice were included in the study, housed in pairs or groups. Testosterone (T) and dehydroepiandrosterone (DHEA) were quantified by radioimmunoassay, corticosterone (CORT) by ELISA. Mean hormone levels were 6.42 pg/mg for T, 23.16 pg/mg for DHEA and 502.1 pg/mg for CORT. Age influenced all hormones by significantly increasing T and DHEA levels and decreasing CORT; only DHEA, significantly higher in grouped mice, was influenced by housing conditions. The influence of age indicates the need for accurate age-related reference intervals, while the higher levels of DHEA in grouped animals suggests that such housing practice may be beneficial for social interactions. In conclusion, it seems that hair hormones quantification may be a good tool for welfare assessment in laboratory mice and may help in refining husbandry.

Difficulties in the molecular diagnosis of Helicobacter rodent infections.

Molecular diagnostic methods using the polymerase chain reaction (PCR) are the gold standard in Helicobacter diagnostics. Most rely on the amplification of parts of the 16S rRNA gene sequence. Therefore, the validity and accuracy of results depends heavily on the PCR design and the time of its publication because new sequences are continually being submitted to databases. Here we report the presence of helicobacter in commercially bred mice supposedly free of this infection. Furthermore, three out of six different commercial laboratories performing helicobacter testing on the same spiked faecal samples failed to detect and identify H. hepaticus. We designed a simple generic PCR assay that amplifies a 261bp amplicon spanning two of the seven variable regions in the 16S rRNA of helicobacter. Using this assay together with an established generic assay designed by Bohr [Bohr, U.R., Primus, A., Zagoura, A., Glasbrenner, B., Wex, T., Malfertheiner, P., 2002. A group-specific PCR assay for the detection of Helicobacteraceae in human gut. Helicobacter 7, 378-383] and then cloning and sequencing their products, we detected the H. hepaticus used in the study that three commercial laboratories failed to detect. We think these assays together could detect all the currently known species of helicobacter and hopefully the new ones as well. In addition, we have been able to identify different species of helicobacter and their relative proportions infecting a single animal. This information has also shown that some helicobacters may have a much broader host range than originally reported.

Mycobacterium gordonae infection in a colony of African clawed frogs (Xenopus tropicalis).

Mycobacterium gordonae is an occasional human pathogen associated with cutaneous infections and nodular granulomatous skin lesions. A case of cutaneous nodular infection caused by M. gordonae in a colony of African clawed frogs (Xenopus tropicalis) is described and confirms this organism to be an opportunistic frog pathogen.

Molecular characterization of a virulent canine coronavirus BGF strain.

Molecular characterisation of a canine coronavirus (CCoV) isolate (BGF), associated with an outbreak of diarrhoea in puppies, showed 92.7% identity with attenuated Insavc-1 strain. Canine coronavirus BGF revealed a full length non-structural protein 3b (nsp 3b), associated with virulence in other coronaviruses, and a highly divergent region at the amino terminal domain of the membrane protein that may be implicated in avoiding the host immune reaction. This new canine coronavirus strain could help to identify virulence factors in coronavirus.

XY female marmoset (Callithrix jacchus).

A marmoset (Callithrix jacchus) with atypical external genitalia was phenotypically and genetically characterized. Testosterone concentration correlated with that of female marmosets. Externally, there was only one opening for the urethra. Internal genitalia were characteristic of those of female marmosets, and consisted of ovaries, with follicles in various developmental stages, and uterus. Microscopically, a normal vaginal structure was found. An XX/XY chimerism and high steroid hormone values are normally found in common marmosets. Genetic analysis was used for in vivo determination of sex. The Y-linked zinc finger protein gene (ZFY) last intron, and sex-determining region Y gene (SRY) exon were found by use of polymerase chain reaction and posterior sequencing analyses, indicating that this marmoset had Y-linked chromosome sequences. Normal SRY exons can, therefore, be associated with female internal sexual organs in marmosets; this may be the first XY female described in non-human primates.

Recommendations for control of pathogens and infectious diseases in fish research facilities.

Concerns about infectious diseases in fish used for research have risen along with the dramatic increase in the use of fish as models in biomedical research. In addition to acute diseases causing severe morbidity and mortality, underlying chronic conditions that cause low-grade or subclinical infections may confound research results. Here we present recommendations and strategies to avoid or minimize the impacts of infectious agents in fishes maintained in the research setting. There are distinct differences in strategies for control of pathogens in fish used for research compared to fishes reared as pets or in aquaculture. Also, much can be learned from strategies and protocols for control of diseases in rodents used in research, but there are differences. This is due, in part, the unique aquatic environment that is modified by the source and quality of the water provided and the design of facilities. The process of control of pathogens and infectious diseases in fish research facilities is relatively new, and will be an evolving process over time. Nevertheless, the goal of documenting, detecting, and excluding pathogens in fish is just as important as in mammalian research models.