Final Report Summary - EUPRIM-NET II (European Primate Network: Advancing 3Rs and International Standards in Biological and Biomedical Research)
Executive Summary:
The new EU Directive on the protection of animals used for scientific purposes (2010/63/EC) foresees various animal protection and welfare measures. EUPRIM-Net II has contributed ideally to some of them through its different activities. For example Article 28 of the Directive requires the Member States to ensure “that staff be adequately educated, trained and competent”. EUPRIM-Net II regularly offered established courses for scientists, veterinarians, technical and animal care personnel on various topics such as General Primate Biology, Behaviour, Diseases and more. A web-based exchange module for veterinarians and other personnel working with NHPs was developed and implemented. European veterinarians were offered specialised training in NHP requirements in a rotation and training program. Last but not least a NHP-specific laboratory animal science course was developed according to the new (2014) FELASA guidelines. Certification of the course is anticipated before the end of this year (2015). This course is now offered without European funding.
“Psychological wellbeing”, allowing the animals to express natural behaviours and gain a measure of control over their environment, is addressed through the field of Animal Behavioural Management (ABM) as well as Positive Reinforcement Training (PRT). Training is now offered in eight different lectures by the EUPRIM-Net ABM/PRT seminar group. What makes this concept unique is that the lectures have been standardised and translated into six European languages meeting the demand for training by local trainers in the mother tongue. The seminar group was supported by research activities to scientifically evaluate the effectiveness of ABM/PRT through behavioural observations by trained ethologists and the assessment of physiological effects.
Knowledge exchange between experts is the basis for advancement in science. EUPRIM-Net II organised a series of workshops and conferences on relevant topics around primate research and keeping, this included events especially targeting the industry as well as non-European institutes in order to maximise outreach.
Although for the time being the total replacement of NHPs in vivo experiments by in vitro models is far from feasible, ethical considerations compel the development of alternative methods. Consequently, with Article 10 the Directive 2010/63/EC “seeks to facilitate and promote the advancement of alternative approaches”, and (the validation of) alternative methods are subject of Articles 46 and 47. EUPRIM-Net II has developed in vitro technologies to replace, reduce and refine NHPs studies within neuroscience/neuroimmunology and vaccine research.
Biobanking is another approach to reduce the number of animals in experiments. Article 27 of Directive 2010/63/EC states that “Member States should, where appropriate, facilitate the establishment of programmes for sharing the organs and tissue of animals that are killed”. The EUPRIM-Net BioBank has provided access to primate materials from different NHP species.
The research projects also developed and improved assays for the health monitoring of NHP. As important refinement cage-based testing systems were developed and advanced, reducing the stress of animals in experiments.
Project Context and Objectives:
The development of alternative technologies promises to replace testing on animals. However, for the foreseeable future until valid alternatives exist, biological and biomedical research as well as toxicological safety testing depends on the use of relevant animal models. In some cases the animal model of choice are non-human primates (NHP). Although this group forms less than 0.1% of all animals used in research, NHP play a special role in providing critical insights that are central for many areas of research owing to their highly developed sensory and cognitive abilities. At the same time research with NHP underlies particularly high ethical (and legal) standards, and a responsible application of the 3Rs concept of Refinement, Reduction, and Replacement is mandatory.
The EU and its national states have instituted a large number of measures to ensure the best welfare of NHP used for scientific purposes. Besides legislative and regulatory efforts (Directive 2010/63/EC on the protection of animals used for scientific purposes) the EU has provided funding to EUPRIM-Net, a network of nine European primate centres from six countries which was established as a Research Infrastructure in 2006 and has now just completed its second round of funding by the Commission (FP7-GA-262443).
In this project the primate centres’ infrastructures and expertise were integrated in order to provide critical services, training and advice to scientific institutions in Europe conducting primate research and to zoological gardens that keep primates. The Network-, Access- and Research Activities all aimed at advancing animal welfare and the 3Rs for NHP in experiments and procedures.
Best Practice in Husbandry and Experimental Procedures. Knowledge exchange and networking is probably one of the most important activities for researchers in order to advance their science and progress. What better occasions can yield opportunities than workshops and conferences on topics in their field? EUPRIM-Net’s aim was to provide a series of workshops on relevant topics all around primate keeping, handling and research. The target audience were primate veterinarians and scientists working in primate research, including the industry and non-European primate centres as important partners and stakeholders.
Veterinarians specialised on non-human primates are rare and require a specialised education before being able to assess the special requirements and needs of NHP. EUPRIM-Net II aimed at contributing to the education of veterinarians in becoming NHP-specialists by giving European veterinarians the opportunity to work hands-on with monkeys. Implementation of an electronic exchange module was aimed to facilitate communication between veterinarians from different primate centres.
ABM/PRT. Behavioural Management techniques and Positive Reinforcement Training (PRT) of laboratory NHPs for participation in procedures has been developed and implemented within EUPRIM-Net to minimise the stress level for the animals, promote more reliable experimental results, and lead to an increased safety, both for animals and personnel. “Psychological wellbeing”, allowing the animals to express natural behaviours and gain a measure of control over their environment is a core aim within EUPRIM-Net. Psychological wellbeing is addressed through the field of Animal Behavioural Management (ABM), involving ethological aspects of captive animal husbandry as well as PRT. In EUPRIM-Net II the aim was to establish an ABM/PRT seminar group in order to spread knowledge and understanding about ABM and PRT of laboratory primates to as many primate facilities as possible in Europe and around the world. The seminar group should be supported by research activities to scientifically evaluate the effectiveness of ABM/PRT through behavioural observations by trained ethologists and the assessment of physiological effects.
Education. Article 28 of the Directive requires the Member States to ensure “that staff be adequately educated, trained and competent”. Therefore, education and training of staff working hands-on with NHP was one of the most important aims of EUPRIM-Net II. For veterinarians and scientists courses were planned on different subjects (modules): General Biology, Behaviour, Husbandry, Diseases and Parasites, Ethics; animal caretakers and technicians have a more difficult time than scientists to find suitable CPD courses. EUPRIM-Net II planned to provide an opportunity for animal caretakers and technicians in primate research to continue their education and improve their skills. For animal caretakers and technicians it may be necessary to provide courses in their mother tongue as they may not be as proficient in English as scientists. To guarantee standardisation and high quality of the courses, certification of the courses by the Federation of European Laboratory Animal Science Associations (FELASA) was aspired.
International Cooperation. In order to maximise outreach EUPRIM-Net aspired to establish a comprehensive exchange programme between important international key players in primate research. EUPRIM-Net aimed to offer a platform of international collaborations linking the centres’ expertise in all relevant areas and resulting in manifold benefits for all participating centres, including EUPRIM-Net.
Co-operation with industrial/commercial partners. In addition to primate centres functioning as public bodies several commercial companies use primates to an extensive degree and thus should be involved in the ongoing discussion and development of the use of primate for research and other purposes. One of EUPRIM-Net II’s objectives was to intensify the collaboration with these industrial partners to exchange and learn from the industry’s experience as well as let them participate in the activities of EUPRIM-Net.
PRIMOCID. With PRIMOCID the objective was to provide access to well-established disease models with the aim to facilitate studies on disease mechanisms and the development of new therapies. The aim was to offer Access to models for rheumatoid arthritis (in rhesus monkeys and marmosets), multiple sclerosis (in rhesus monkeys and marmosets), allograft rejection (skin, in rhesus monkeys), and (new to this project) to infectious disease models and neurodegenerative diseases. Projects aimed at the development of alternatives for animal testing, such as organotypic cultures or refinement of existing models was appreciated. Development of new disease models was also possible, but on a limited scale as this should fit within the limitations for access provided with regard to time and budget. In addition to the disease models access was to be given to the primate laboratories at the BPRC and the possibility to study primate behaviour.
BioBank. Tissue and genetic material obtained from primates represents a valuable resource as it can be used in specific studies in biomedical research. However, the provision of primate material is difficult and time demanding and hence, comparative studies involving primate research are often challenging or even impeded due to limited or non-availability of appropriate material. The aim of EUPRIM-Net II was to make available collections of diverse primate materials in three institutes of EUPRIM-Net (DPZ, BPRC, WWU) both for internal use and for external scientists. The idea behind the BioBank is to ensure the sharing of rare and valuable primate resources through more efficient access to specific types of samples available to researchers for biomedical research as well as for conservation studies. The EUPRIM-Net II BioBank aimed to: 1) accelerate progress in the solution of biomedical and other scientific problems that depend on supply of primate biological materials; 2) contribute to the 3R-concept (reduce, refine, replace) by reducing the numbers of primates needed through efficient resource sharing; 3) substantially enhance various European research fields through access to restricted primate materials, such as non-invasively collected DNA samples from endangered species, RNA and cDNA samples from a variety of healthy and diseases tissues; 4) provide genetically characterized and, therefore, certified primate material. The EUPRIM-Net II BioBank consists of: A) a tissue bank representing both normal and diseased tissues of primates including blood and serum samples (DPZ, BPRC, WWU); and B) a gene bank with DNA, RNA, cDNA.
Tools to Measure Infection and Diseases. Further EUPRIM-Net aimed to develop and apply new technologies for the detection and validation of diagnostic and prognostic markers of viral and bacterial diseases in NHP. The specific objectives were: 1) to develop multiplex assays for detection of pathogens, and for the determination of immune responses and cytokine levels; 2) to develop transcript-analysis assays for the identification of diagnostic and prognostic RNA markers of disease progression; 3) to explore the use of non-invasive, or less-invasive sampling in these assays, 4) to standardise protocols and exchange data between partners.
The implementation of the new technologies in NHP research has a high merit to strongly contribute to the 3R principle. Use of archival tissue samples, or non-, or less-invasively obtained samples further contributes to the 3R’s. Importantly, all techniques established in course of this research project can easily be adapted and applied to other pathogens or diseases of NHP. The techniques developed in this Research Activity will have considerable impact on future research in the EU involving NHP. This will not be limited to the field of infectious diseases, as they can be widely used in research on animal models for human diseases.
Cage-based Systems. Recording of physiological data, especially invasively, but also non-invasively, often requires that animals are handled and restrained in their movements during the experimental procedures. In system neuroscience, for example, constraining the animal’s head movements is the standard procedure for microelectrode recordings from the animal’s brain, while the animal performs cognitive tasks. The interaction with the animal bears the potential to induce stress, which should be avoided, might be detrimental to the behavioural learning progresses, and can have confounding effects on the physiological parameters in question. If physiological data is not collected for research purposes, but to monitor animal welfare in a colony, such stress leads to the paradoxical situation that attempts to optimize animal welfare might actually reduce it. The objective of this research activity was to develop and implement procedures for remotely monitoring physiological and behavioural data from monkeys in their housing environments. Telemetry systems were combined with behavioural test systems for integrated and automated data collection. The expected impact is a considerable reduction of potential stress for monkeys in experimental situations, and improved possibilities to continuously monitor animal welfare. These developments help to identify sources of stress and to reduce stress in research monkeys. Induced stress is a confounding factor for many research questions. Reduction of stress contributes to the refinement of studies by improving the quality of the collected data. As a consequence the number of animals needed for a study is reduced and animal welfare in experimental conditions is improved.
In vitro Technologies (Alternative Methods). Another important aim of this project was to develop and apply new in vitro technologies leading to Replacement, Reduction and Refinement (the 3Rs) regarding the use of NHP in biomedical research. The specific objectives were: 1) to develop in vitro model systems for research on the central nervous system; 2) to develop in vitro test methods for vaccine research.
Studies on the CNS that use NHP often go accompanied with moderate to severe discomfort for the animals. This is often due to the invasive nature of the studies or to the very long preclinical incubation times of e.g. transmissible neurodegenerative diseases. Moreover, the negative public perception of CNS studies provides an extra motivation for the development of alternatives. The development and validation of in vitro primary cell cultures, transgenic cells and specified cell culture systems can provide researchers with the possibility to predict the outcome of in vivo experiments and to screen compounds or therapeutics that directly target resident CNS cells as a pre-in vivo test phase, reducing the number of NHP needed for in vivo studies. The development and validation of slice culture techniques can be used to address even more complex research questions, further reducing animal numbers for in vivo studies.
In HIV vaccine research, controlled prime-boost regimen strategies followed by experimental exposure (challenge) to simian or human/simian immunodeficiency viruses (SIV or SHIV) is, as yet, only feasible in NHP. The protective outcome of SIV vaccination can only be measured in vivo, leading to studies where NHP are kept under experimental conditions for extended periods of time. The aim was to develop and validate in vitro methods to predict the clinical outcome of vaccination ex vivo, ideally rendering the experimental in vivo challenge with virus unnecessary leading to a considerable reduction of the duration of in vivo experiments. One of the major obstacles in the development of vaccines is the selection of potent adjuvants that are safe and licensed for human application. Adjuvants and vaccine formulations are tested in various animal models, including NHP, causing significant degrees of discomfort in the form of unwanted side-effects. The objective was to develop and employ in vitro technology to select and develop new adjuvants with good adjuvating potential, but with minimal side-effects. Although this project contributes mainly to refinement, the topic is so general and involves so many animals that even slight improvements will generate considerable effects regarding animal welfare.
Project Results:
Best practice and harmonisation in husbandry and experimental procedures with NHP. Knowledge exchange and networking is probably one of the most important activities for researchers in order to advance their science and progress. To this end, twelve workshops were organised targeting veterinarians and scientists and one also addressing regulatory authorities as well as the public. Connections to other European organisations focusing on non-human primates [European Primate Veterinarians (EPV)] and professional animal training experts [AnimalConcepts] were exploited. The NHP-using industry and non-European primate centres are valuable exchange- and collaborative partners, thus five workshops were especially targeted towards these groups. One workshop in Nairobi, Kenya included a minisymposium that was attended by the German Ambassador in Kenya. All in all the workshops were visited by more than 400 participants.
EUPRIM-Net II workshops between 2011 and 2015 – brief summaries:
“General techniques and best practice using NHP”: This workshop was co-hosted by EPV and focused on minimal invasive techniques to be used in both research centres and zoos. Besides presentations and discussions, training on endoscopic techniques by the experts in a “dummy system” and more detailed information on thermographic techniques were provided.
“Primate Welfare & Training Workshop”: A summary of the workshop has been published in “Enrichment Record” with both Euprim-Net members (Marit Vernes) and AnimalConcepts (Sabrina Brando) as co-authors. [Brando S, Vernes M (2014). Primate Welfare and Training Workshop. Enrichment Records, Winter 2014]
“Food-and-fluid control”: The workshop was co-hosted by AnimalConcepts; the aim was to share knowledge on training methods to refine the use and care of NHP in neuroscience research. Veterinary care of these animals, the possibilities to prevent or diminish food and fluid control and behavioral management were discussed.
“Primate nutrition and its implications in research and on animal health”: The workshop was co-hosted by EPV and included effects of primate nutrition on various aspects of animal welfare and behavioral management. In addition, several case reports discussing practical aspects were presented and discussed. These discussions included topics on food supplements, food restriction, obesity, and skin problems.
“Workshop on the microbiome and animal health”: A one-day workshop on the microbiome and animal health, specifically aimed at non-human primates. The discussions included practical aspects on analysis and more detailed effects of changes in the microbiome and health aspects.
“Non-invasive imaging in non-human primates”: A workshop on non-invasive imaging in non-human primates was conducted including Refinement of infectious disease studies using advanced imaging; SPECT in non‐human primates; Optical imaging in preclinical research; Hyperpolarized MRI – Potential and perspectives; Structural and functional cardiac imaging; Research in non‐human primates – role of in vivo imaging; Anatomical and functional MRI in awake marmosets; Diffusion weighted imaging: Basic concepts and recent technological advances; Arterial spin labeling: Non‐invasive perfusion measurements and beyond; Neurotransmission and brain metabolism - Advanced magnetic resonance spectroscopy.
“Methods and Practices for professional banking of tissues and material”: One of the main issues was the visibility and how to reach the general researchers. From this point of view, much input was provided by the users and the external speakers.
“Alternative methods for the use of non-human primates in biomedical research”: The workshop included lectures by key scientists in the field of alternatives as well as by experts from governmental and non-governmental organizations. Furthermore, parallel sessions were organised to stimulate discussion on the challenges of advancing the use of alternative methods for NHP. Subgroups voted on four statements and together composed a list with opportunities and priorities. [ALTEX. 2014;31(4):520-9. doi: http://dx.doi.org/10.14573/altex.1406231(se abrirá en una nueva ventana). Epub 2014 Jul 24.]
Workshops conducted together with industrial and/or non-European partners:
“Positive Reinforcement Training (PRT) and Animal Welfare“ and “Practical training and implementation of environmental enrichment”: These two workshops were conducted at the Institute of Primate Research (IPR) in Nairobi, Kenya. The Basics of PRT were taught and the possibilities of implementing enrichment practically demonstrated. One of the workshops was accompanied by a minisymposium attended by the German Ambassador.
“Novel Insights into Primate Genomics: Impact for Biomedical Research“: This workshop was initiated by our industrial partner and co-hosted by EUPRIM-Net because species selection and suitability of an individual experimental animal may depend on the genetic background, which may not always be known. [Trends Genet. 2014 Nov;30(11):482-7. doi: 10.1016/j.tig.2014.05.004]
“Refinement of non‐human primate use in research“: This workshop was initiated by our industrial partner as satellite workshop at the symposium of the International Primatological Society and co-hosted by EUPRIM-Net because an important part of this workshop was on education of staff working with NHP.
“Workshop on the use and the advantages of marmosets in translational biomedical research”: Genetically modified marmosets most likely will become exceptionally relevant in academic and industrial research in Europe, the US, and Japan. However, since important biological characteristics of this NHP species may be affected in genetically modified marmosets, it is of utmost importance to proactively consider possible effects of the modifications on the monkeys’ well-being and housing. In this context, the 3R (Reduction, Refinement, Replacement) and 4R (3Rs plus) principles were discussed.
Education & Training. Article 28 of the Directive requires the Member States to ensure “that staff be adequately educated, trained and competent”. To this end one of the most important aims of EUPRIM-Net II was to provide education to staff working hands-on with primates, including all occupations involved from animal care takers, animal trainers and technicians to scientists and veterinarians. Participants came from ten different European countries and four non-European countries.
Animal care takers, animal trainers, technicians:
This group has a more difficult time than scientists to find suitable Continued Professional Development (CPD) courses. EUPRIM-Net II provided a highly appreciated opportunity for animal caretakers and technicians in primate research to continue their education and improve their skills. For them it may be necessary to provide courses in their mother tongue as they may not be as proficient in English as scientists. This was taken into consideration. More than 125 participants took part in six courses carried out in Germany and the UK. Content of the courses were general primate biology, diseases and parasites, husbandry, handling, and its challenges (e.g. zoonses), communication of primate research (problems, strategies and suggestions).
Veterinarians:
The rotation system for training of veterinarians and assistants/technicians was planned to foster species-specialisation of veterinarians with regard to non-human primates. The programme admitted six veterinarians who were trained in two-week training visits at the BPRC visits. After each training period, participants received a training certificate.
Veterinarians and scientists:
Ten CPD courses were offered to veterinarians and scientists that were in total attended by 210 participants. Course contents were offered in modules on general biology; primate sociobiology, husbandry, and nutrition; primates in biomedical research; primate husbandry, medical aspects, security, advanced methods and ethics; general primatology, behaviour, cognition and neurophysiology; marmosets as animal models. The course language was English or French.
In the beginning of 2014 FELASA (Federation of European Laboratory Animal Science Associations) revised their recommendations for the accreditation of education and training courses in laboratory animal science. According to the new system it became possible to introduce species-specific courses that could be certified by FELASA. This opened up the way for EUPRIM-Net II to develop primate–specific Laboratory Animal Science (LAS) courses according to FELASA guidelines, function A+B, the highest recognised certification in Europe and address Continued Professional Development as mentioned in Annex I. The first course was held within the term of EUPRIM-Net II. This course was divided in two parts, encompassing an e-learning phase that was specifically developed for this course as well as practical parts. The accreditation process of the primate-specific Laboratory Animal Science course is ongoing beyond the term of EUPRIM-Net II. Nevertheless, development of this course would not have been possible without the support of the EU Commission.
All staff working hands-on with monkeys:
A Seminar Group has been formed, consisting of veterinarians, ethologists, and animal trainers. Together, this group has created eight lectures on topics regarding Animal Behaviour Management (ABM), ranging from breeding laboratory primates to enrichment and problem solving. The group now offers to come to facilities keeping NHP and present these lectures as in-house training. They can customise a combination of lectures according to the needs of the facility; for instance, the facility might be interested in learning more about how to conduct behavioural studies and how to start training their primates. All lectures are 3-4 hours long and the target audience is animal technicians; however all personnel involved in managing the colony, including scientists, will benefit from attending. During the EUPRIM-Net II term (2011-2015) 19 seminars with one or more lectures were given to NHP-keeping facilities within and outside Europe of which only a basic number was subsidised by the EU; since then the in-house trainings have become well-known and popular and requests have already come in for the time beyond EU-funding for EUPRIM-Net II. A short description of the different lectures can be found below.
Lectures offered by the ABM/PRT Seminar Group (www.euprim-net.eu/network/ABM-seminars.html):
Ecology and behaviour of primates - In this lecture natural including social behaviour of common laboratory primates is described - important knowledge when housing a particular species.
How to optimize laboratory primate housing and cages - An empty cage is useless to the animals. This lecture gives ideas of how to plan and improve the animals' living quarters. Improved welfare may improve the quality of the science!
An introduction to primate training - This lecture gives the basic tools to start training animals to collaborate in procedures, facilitate housing and handling, and improve human-animal relationships!
Environmental enrichment in primate facilities - Why and how a modern enrichment program may be implemented is presented in this lecture in a structured approach.
Breeding laboratory primates - How can breeding success be optimised? This lecture gives ideas with regards to primate breeding, incorporating veterinary, behavioural and practical perspectives.
Solving behavioural problems - Animals don't always behave the way we want them to. In this lecture, structured approach is presented to solving common behavioural problems in primate facilities.
How to observe and measure behaviour - Did the enrichment programme work? Which individuals are compatible for pair housing? From a management perspective observing and measuring behaviour is capital. This lecture adresses those issues.
Why stress matters - In this lecture, we argue that we need to be concerned with reducing stress both to improve welfare and the quality of the science.
Outreach & Dissemination. The EUPRIM‐Net consortium takes serious interest in providing the public community with solid information about the keeping and wellbeing NHP used for research purposes in the primate centres of the Infrastructure and their benefit for health research. This is especially important in the current setting, where recently various ways (European citizens’ initiative, European Parliament resolutions, petitions, and parliamentary questions) have been attempted to call into question the value of Directive 2010/63/EU, to try and repeal it and ban the use of animals in research and development.
To this end factual reporting and information sharing on research with and on NHP is more crucial than ever. Therefore, a virtual tour was designed and developed that was regarded an appropriate approach to inform a broad audience about the breeding and housing facilities and the way non‐human primates are bred, kept and handled for research purposes in primate centres. For the virtual tour (www.dpz.eu/en/info‐center/mediacenter/virtual‐tour.html) the premises of the German Primate Center (DPZ) were chosen as an applicable example also reflecting the work of the other EUPRIM‐Net institutions.
BioBanking. Over the recent years the value of biobanking and its contribution to the 3Rs (replacement, reduction, refinement) has become more and more obvious. This has been recognised by European legislation and put in writing with Directive 2010/63/EU on the protection of animals used for scientific purposes: Article 18 urges member states to facilitate where appropriate, the establishment of programmes for the sharing of organs and tissues of animals killed. And in fact one such programme has been established by the EU‐funded infrastructure EUPRIM‐Net with the EUPRIM‐Net BioBank.
Over its 54 months duration EUPRIM‐Net II has provided 97 organ‐ and tissue samples of non‐human primates to 21 research projects. Since maximally 20% of the total infrastructure can be subsidised by the EU that means that at least 485 organ‐ and tissue‐samples have been distributed by the EUPRIM‐Net institutes involved. It is actually more likely that all in all close to 600 samples have been provided by the consortium over the EUPRIM‐Net II duration, as one institute was not involved in the EUPRIM‐Net BioBank at the time of proposal submission but since then have established a biobank and have distributed non‐human primate samples. One institute was involved but did in fact not distribute samples via the EUPRIM‐Net biobank due to the complexity of the administration.
The number of samples distributed via the EUPRIM‐Net BioBank reflects the ratio of non‐human primates versus other species, especially rodents, used for research purposes. Considering this niche the number of distributed samples is actually quite impressing, especially with respect to the 3Rs and the number of animals which may have been spared. The research projects that were provided with organ‐ and tissue samples were in the fields of immunology, oncology, endocrinology, neuro‐ and eye anatomy, neurophysiology, drug screening, genetics and epigenetics, and HIV research.
Gene samples from non‐human primates are rare and yet a variety of scientific disciplines can make good use of them. Whether used in research on conservation issues, evolution and epigenomics, genetic backgrounds of social behaviours or for conducting medical research like finding cures for diseases like B‐cell‐malignancies or HIV: DNA or RNA of different NHP species, from Rhesus or Cynomolgus macaque to chimpanzee and bonobo from the EUPRIM-Net II BioBank have contributed to scientific progress during the past 54 months and were much appreciated by the scientists.
All in all, the EUPRIM‐Net Genebanks provided 80 samples that were funded by the European Commission via EUPRIM‐Net II to 17 research projects. As maximally 20% of the total infrastructure can be reimbursed by the EU that means that over the 54 months of EUPRIM‐Net II funding at least 400 gene samples were provided to the European scientific community.
PRIMOCID. With PRIMOCID (Non-Human Primate Models of Chronic Immune Disorders) Transnational Access was provided to eight research projects, supporting scientists from six different European countries with experimental animals and expertise. The projects were in the fields of Multiple Sclerosis, Tuberculosis, Malaria, Alzheimer Disease, DNA vaccines, arthritis, and behaviour.
Tools to measure Infection and Disease in NHP. The 3R concept implies that more data are obtained from minimal numbers of animals, and from small samples that are preferably collected using non- or less-invasive sampling techniques. To this end several assays have been developed in EUPRIM-Net II:
When working with NHP, there is always the possibility of anthropo-zoonotic transfer from humans to NHP and vice versa. To better assess the risk of infection a multiplex assay for the detection of respiratory pathogens in NHP was developed and validated. Using this assay, infections with respiratory viruses could be detected in colonies of NHP. For the direct detection of different viruses, a Luminex-based multiplex assay, the so called Respiratory Luminex assay (RLA), was developed and validated. The same technology platform has been used to identify cytokine signatures in materials from macaques infected with Mycobacterium tuberculosis (Mtb) and evaluate their potential to provide diagnostic indicators.
Characterisation of the gut microbiome from the colony of healthy rhesus macaques at the BPRC was started and feces of animals of different age groups and sex were sampled. Libraries of 16S-rRNA were prepared and further sequenced. At least three new bacterial species were identified and have been or will be submitted to the German Collection of Microorganisms and Cell Culture (Leibniz-Institute DSMZ) for further in-depth characterisation and inclusion in their collection. Currently, a new Peptococcus strain has been identified in the rhesus macaques (proposed name: Peptococcus simiae), and has been submitted to this collection. Several other new bacterial strains will also be sent out to the DSMZ for in depth analysis soon. This work will be continued to better understand the reciprocity of the immune system with the microbial community of the gut.
Alternative approaches to dissecting immune responses in GBV-B-infected tamarins were explored (GBV-B is the tamarin counterpart of the hepatitis C virus). The GBV-B detection system was improved by using an extensive archive of a range of biological materials from naive and infected animals as well as new animal studies where appropriate.
More assays were developed for the identification of diagnostic and prognostic markers of viral and bacterial diseases in NHP. These apply transcript analysis and have a potential to improve health surveillance programs in NHP-keeping facilities. E.g. MPXV-regulated miRNAs could be detected in samples from infected macaques and cell cultures. Moreover, Herpes B Latency associated transcript (LAT) could be detected and eight new miRNAs identified in herpes B virus (BV)-infected cells. Complementing this assay, a quantitative Polymerase Chain Reaction (qPCR) assay for the detection of BV-specific nucleic acids in ganglia of rhesus macaques could be established. A peptide microarray technique was used to develop an assay to measure antibodies against the two immunodominant glycoproteins B (gB) and D (gD). This should avoid “false negative” results possibly obtained by the commercially available HSV-1-ELISA.
Dedicated mRNA signatures from Mtb-infected macaques could be validated that are prognostic RNA markers of disease progression and miRNAs associated with the pathology of viral hepatitis were explored as a result of GBV-B infection in tamarins.
Non-invasive or less-invasive sampling for use in multiplex or transcript-based assays was explored and the development of non-invasive sampling procedures for routine virus surveillance was initiated. This work has not been completed; nevertheless, in total more than 400 swabs have been collected from the BPRC colony animals and stored at -80°C.
Proof of concept was established for the use of dried blood spots for evaluation of cytokines using a Luminex multiplex immunoassay. Exploring the use of non-invasively sampled materials for the detection of miRNA has started. Literature suggests that miRNA can be detected in urine, and thus urine samples taken at termination have been stored in order to perform comparisons with assays undertaken on serum and liver when these are fully analysed.
Cage-based wireless recordings in unrestrained monkeys. Recording of physiological data, especially invasively, but also non-invasively, often requires that animals are handled and restrained in their movements during the experimental procedures. The interaction with the animal bears the potential to induce stress, which should be avoided, might be detrimental to the behavioural learning progresses, and can have confounding effects on the physiological parameters in question. If physiological data is not collected for research purposes, but to monitor animal welfare in a colony, such stress leads to the paradoxical situation that attempts to optimise animal welfare might actually reduce it. The research reported here had two major goals. First, comparison of stress levels in different experimental procedures in primates, particularly testing for the effect of a cage-based behavioral testing approaches compared to approaches which require handling the animals; second, development of a cage-based behavioral testing and training procedures for rhesus monkeys suitable for neuroscientific research. The major finding of this reporting period and WP9 as a whole supports a cascade of stress level increments one may have expected: Cage-based testing can have an enrichment effect in socially healthy groups in housing colonies of baboons, which may be considered to be the least stress imposing setting investigated here. How a cage-based test system in a lab environment and in different species compares to that remains to be investigated. In a laboratory environment, using cage-based approaches compared to lab-based approaches including animal handling, in marmoset monkeys preliminarily show lower acute stress responses in cage-based approaches compared to lab-based approaches. Yet, lab-based approaches with regular handling in marmosets did not lead to sustained increased stress levels which would show in sleep or other physiological stress-associated parameters measured remotely. Results were partly obtained from different NHP species and may not transfer to other species. But if cage-based approaches also reduce stress in other NHP species and settings then the cage-based behavioral testing and automated training procedure developed for rhesus monkeys in neuroscientific context in EUPRIM-Net II could mark a first step towards a refined experimental approach in this field.
In vitro technologies to replace, reduce, and refine NHP studies. For the time being biological and biomedical research as well as toxicological safety testing depends on the use of relevant animal models. In some cases the animal model of choice are NHP. Nevertheless, the number of animals used in experiments ought to be as small as possible. To this end EUPRIM-Net II has conducted research to develop in vivo technologies that can deliver (preliminary) results necessitating fewer NHP in the experiment.
This research was able to validate primary in vitro glia cell cultures as pre in vivo screening method. Some tissue-specific findings in this research project might be therapeutically exploitable in neuro-inflammatory and neurodegenerative diseases. Furthermore organotypic brain slice cultures from rhesus macaques were characterised: Although these slices were demonstrated to be responsive to neuro-inflammatory stimuli, exposure to neurotoxic agents resulted in massive cell death and general loss of viability. Whereas the organotypic slice cultures thus represent a valuable new tool to study (innate) neuro-inflammatory responses, their use as models for neurotoxicity is limited.
For the study of BSE and transmissible neurodegenerative diseases in NHP in general, there is a lack of an appropriate in vitro system. The strategy was the establishment of transgenic rabbit kidney (RK13) cell lines which overexpress full-length prion protein. Established transgenic cell lines expressing PrPc of either squirrel monkey (Saimiri sciureus) or cynomolgus monkey (Macaca fascicularis) BSE did support prion propagation and therefore could not be used for the diagnosis of macaque-adapted BSE; however RT-QuIC could be established and adapted for the ultrasensitive detection of prion activity in various specimens. With this new technique prion replication in peripheral tissues of BSE-inoculated macaques can be assessed. The detection of prions in different clinical specimens (e.g. CSF, lymph nodes, blood), that can be obtained routinely and/or by mildly invasive methods and even before the occurrence of clinical manifestations is of high importance. Those clinical specimens in combination with ultrasensitive methods, like RT-QuIC, can be used to predict disease outcome in vivo thereby significantly reducing the time necessary for the keeping of prion-inoculated macaques. We therefore consider the establishment of RT-QuIC for specific and highly sensitive detection of macaque-adapted BSE as equivalent to the anticipated deliverable to produce BSE-susceptible cell lines.
Although infection rates are declining due to improved education and enhanced worldwide access to antiretroviral medication, HIV-infection remains a global health problem. However, an AIDS vaccine is still lacking and NHP research remains a necessity to reach that goal. An in vitro test that delivers early prediction of AIDS vaccine efficacy in human or macaques, which represent the major species for preclinical AIDS vaccine trials, could contribute to vaccine development as well as to refinement and reduction of studies conducted in NHP. We therefore aimed at developing predictive in vitro protocols in the monkey model of AIDS. To do so, we primarily analysed samples from three different experiments. Extending the original application, we used four approaches to refine the macaque model of AIDS: 1) We validated and/or detected other prognostic marker for the macaque model of AIDS; 2) As a potential additional prognostic marker, we quantified expression of innate immunity genes and identified correlates of protection that can be used to determine AIDS vaccine efficacy ex vivo within 48 h post vaccination. 3) We found that a single nucleotide polymorphism (SNP) in DC-SIGN/CD209 - that is similar to a human SNP - can be used as a further prognostic marker to distribute animals evenly into the various experimental groups (as means of standardization) to reduce number of experimental animals without losing statistical significance (manuscript in preparation). 4) We investigated antiviral non-cytolytic activity of CD8 cells in an attempt to identify a novel prognostic marker that can serve as standardization of experiments. While we did not find strong evidence that this activity influenced susceptibility to SIV-infection, candidate genes linked to this activity were detected by transcriptional profiling.
Adjuvants are formulations, which upon administration lead to non-specific immune stimulation. They are used to induce immune responses directed against pathogens (as for vaccination purposes) or to generate immune responses against components of the body itself (as in experimental animal models of human auto-immune diseases like e.g. multiple sclerosis, diabetes or rheumatoid arthritis). However, some of the more potent adjuvants are notorious for their adverse effects. Complete Freund’s Adjuvant (CFA) for example is known for the development of granulomatous skin lesions, hampering application in humans and causing various degrees of discomfort to NHP. There is therefore an urgent need for new and safe adjuvants for human use and a particular need to develop new, cleaner adjuvants for use in experimental animals. With the recent discovery of a family of molecules that form part of the innate immune system, the Toll-like and NOD-like receptors (TLR and NLR resp.), important progress was made in our understanding of how adjuvants work on a molecular level. Upon activation these receptors deliver a potent non-specific activating signal to the immune system, much like adjuvants do. We have used this knowledge to develop in vitro systems to model the adjuvating effects, in the form of TLR and NLR bioassays, as well as in vitro systems to model the adverse effects, in the form of in vitro granuloma models. Both assay types have been developed and extensively used to both identify new adjuvant candidates with good adjuvating potential as well as to characterise the granuloma-inducing potential of new adjuvant candidates.
Moreover alternative approaches to CFA have been developed using mycobacterial strains that specifically lack the expression of sets of proteins inducing less lung granulomas than CFA. Second, as a bottom-up approach, we have characterised the overall innate immune responses evoked by M. butyricum, both qualitatively and quantitatively, and used this information to compose an entirely new adjuvant (MiMyc) that mimics these responses.
Scientific evaluation of Animal Behavioural Management (ABM) and Positive Reinforcement Training (PRT). Research was carried out within EUPRIM-Net II to investigate effects of ABM/PRT measures or experimental refinement methods. In this context the impact of voluntary cognitive testing on captive group-housed long-tailed macaques’ (Macaca fascicularis) stress level and social behavior was examined. There are two opposing views on the potential impact of experimental testing: Separation from the group and/or frustration during the experiment might cause an increased stress level. Alternatively, experimental testing might serve as enrichment for captive individuals and thus contributes to the animals’ welfare. When data collection will be finished (September 2015), we will pool the data to enhance sample size for the planned paper “The impact of cognitive testing on stress levels in captive long-tailed macaques (Macaca fascicularis)”. Overall, the results strongly suggest that cognitive testing is neither detrimental to the participating individuals nor to the group as a whole, in terms of agonistic interaction rates.
Moreover decision making in animal trainers was analysed as a function of training frequency and a paper finalised that has recently been submitted for publication. A textbook chapter was written on laboratory primate training that will shortly be submitted for publication in an online, open access format. During EUPRIM-Net II at least seven papers and five additional manuscripts in the field of ABM/PRT were produced in different stages of publication. Note that two papers also explore intricate uses of negative reinforcement training (NRT) in combination with PRT. An interesting finding was that NRT may indeed be used to reduce fear and give animals more control over their situation. Data collection has started on the impact of early socialization on laboratory primates. This project has stalled but we’re hoping to relaunch it during 2016. Blood cortisol data was analysed from animals in different stages of training. However, the results were inconclusive and we’ve decided not to pursue a publication elaborating on that issue.
Potential Impact:
For the foreseeable future until valid alternatives exist, biological and biomedical research as well as toxicological safety testing depends on the use of relevant animal models. In some cases the animal model of choice are non-human primates (NHP). Understanding the biology of nonhuman primates helps us to understand our own biology. That is why NHP play an important role in biological and biomedical research. This group of animals provides models which are essential for research in genomics and biotechnology for health. The successful development of new strategies against human diseases like infectious (e.g. HIV, hepatitis) and neurological diseases (e.g. Alzheimer´s or Parkinson Disease) or cancer depends on the availability of NHP or biological material of NHP origin. This also holds true for the development of new therapeutics (vaccines, gene therapy) and transplantation research. Although this group forms less than 0.1% of all animals used in research, NHP play a special role in providing critical insights that are central for many areas of research owing to their highly developed sensory and cognitive abilities. At the same time and for the same reasons research with NHP underlies particularly high ethical (and legal) standards, and a responsible application of the 3Rs concept of Refinement, Reduction, and Replacement is mandatory.
This is especially important in the current setting, where recently various ways (European citizens’ initiative, European Parliament resolutions, petitions, and parliamentary questions) have been attempted to call into question the value of Directive 2010/63/EU on the protection of animals used for scientific purposes, to try and repeal it and ban the use of animals in research and development. To this end factual reporting and information sharing on research with and on NHP is more crucial than ever.
The EU and its national states have instituted a large number of measures to ensure the best welfare of NHP used for scientific purposes. Besides legislative and regulatory efforts (Directive 2010/63/EU) the EU has provided funding to EUPRIM-Net, a network of nine European primate centres from six countries which was established as a Research Infrastructure in 2006 and has now just completed its second round of funding by the Commission (FP7-GA-262443).
Outreach. With EUPRIM-Net II the EU has continued support to a virtual European Primate Centre where standards are high, best practices are used and all NHP-keeping facilities in Europe and worldwide can benefit. Owing to their highly developed sensory and cognitive abilities, it is important to exchange and network for the welfare of the NHP in experiments. If the experimental animal is stressed this may have confounding effects to the physiological parameters in question and thus detrimental to the advancement of biological and biomedical research. EUPRIM-Net II facilitated networking and mutual knowledge transfer by offering high-end workshops on significant topics around the keeping of NHP for research purposes or advanced technology in primate research, not only for its consortium members, but also outreaching to industrial stakeholders and researchers outside Europe. Important collaborations have been initiated that show how important animal welfare issues are also to these groups and joint efforts will contribute to advancement of animal welfare and thus biological and biomedical science beyond the state of the art. Last but not least the workshops provided opportunities for exchange on state-of-the-art technologies, contributing on the best research with NHP in Europe.
EUPRIM-Net II not only addressed scientists but outreached also to non-professionals. This is of particular importance because for the common public it may be difficult to understand why and how NHP are used in experiments and to what extent they may suffer distress during procedures. In the virtual tour through a European Primate Centre that has been produced by EUPRIM-Net II one can learn about research with NHP and how the animals are kept, with the aim to increase acceptance of NHP research among European citizens (http://www.dpz.eu/en/info-center/media-center/virtual-tour.html(se abrirá en una nueva ventana)).
Especially in times of European Citizen’s Initiatives and Petitions against research with animals, it is important to disseminate solid information on NHP research and how animals are kept and handled. Owing to this climate, EUPRIM-Net II has hosted a workshop “Alternative methods for the use of non-human primates in biomedical research” bringing together key scientists working on alternatives to animal research with experts from governmental and non-governmental organisations. During this workshop parallel sessions were organised to stimulate discussion on the challenges of advancing the use of alternative methods for NHP. Subgroups voted on four statements and together composed a list with opportunities and priorities regarding implementation of all 3Rs alternatives, stem cells, NHP in vivo models, and communication. The white paper that resulted from of the Workshop [ALTEX (31)4: 520-529; DOI: http://dx.doi.org/10.14573/altex.1406231](se abrirá en una nueva ventana) can serve as guiding document for future activities.
Education. Article 28 of the Directive on the protection of animals used for scientific purposes (2010/63/EC) requires the Member States to ensure “that staff be adequately educated, trained and competent”. Even before the revision of the European Directive, EUPRIM-Net II had designed a strong programme for continued professional development of staff working with and on NHP. Workshop-like courses on different topics with international experts offered high-end education for scientists and veterinarians, while moreover courses were offered in their local language to animal trainers and care takers as well as technicians, in many instances a rare opportunity for them to receive customised education. Moreover, these courses have represented a very important opportunity for Continued Professional Development (CPD) for all 335 participants from ten different European and four non-European countries and have given NHP staff a chance to exchange with colleagues without the fear of resentments which they may encounter at mixed events.
Developed from scratch was a NHP-specific course meeting FELASA (Federation of laboratory animal science associations) guidelines for the accreditation of laboratory animal science (LAS) courses. This course comprises theoretical and practical contents and a comprehensive E-Learning part was developed especially for this course. The course will probably be accredited before the end of this year (2015) and will then be the first NHP-specific course in Europe certified by FELASA, revolutionising basic education of new staff working with NHP and contributing to harmonisation of NHP-staff education in Europe.
Thirdly, a EUPRIM-Net Seminar Group was formed with participation of ethologists, veterinarians, animal trainers and scientists who created and now offers eight standardised lectures on different topics around NHP keeping and handling to NHP-keeping facilities as in-house trainings (www.euprim-net.eu/network/ABM-seminars.html). These trainings are extremely important because the EUPRIM-Net expert inspects the on-site conditions and can customise the training according to the needs of the facility. Even beyond the EUPRIM-Net II term, Seminar Group members have been asked to give trainings to academic and industrial laboratories.
BioBanking has a high merit for the 3Rs as the sharing of tissues and organs implies the reduction of experimental animals sacrificed for scientific purposes. Over its 54 months duration EUPRIM‐Net II has provided 97 organ‐ and tissue samples of NHP to 21 research projects. All in all, the EUPRIM‐Net Genebanks provided 80 samples that were funded by the European Commission via EUPRIM‐Net II to 17 research projects. Since maximally 20% of the total infrastructure can be subsidised by the EU that means that at least 485 organ‐ and tissue‐samples (actually, close to 600 samples are more likely) and at least 400 gene samples have been distributed by the EUPRIM‐Net institutes involved. These samples have contributed to progress of European research, at the same time sparing animals that would otherwise have been sacrificed especially for the research project in question. Apart from the number of distributed samples it should be emphasised that the EUPRIM-Net BioBank is a unique and valuable resource in Europe for access to characterised, high quality NHP biomaterial used by researchers in biological and biomedical research accelerating progress in the solution of biomedical and other scientific problems that depend on supply of NHP biomaterial.
PRIMOCID. In some cases NHP is the only appropriate animal model that can give answers to specific scientific questions. Experimentation with NHP requires expert knowledge and specialised facilities. Through PRIMOCID (Transnational Access to Non-Human Primate Models of Chronic Immune Disorders) the infrastructure project EUPRIM-Net II has offered expert knowledge, facilities and animals to European researchers. Over the 54 months eight projects have been funded by the European Commission. Since maximally 20% of the total infrastructure can be subsidised that means that at least 40 excellent projects in the fields of Multiple Sclerosis, Tuberculosis, Malaria, Alzheimer Disease, DNA vaccines, arthritis, behaviour and more have been conducted that would not have been possible without the kind of expert service offered by the EUPRIM-Net consortium, or only under conditions not optimal for the research or the animals.
Tools to measure Infection and Disease in NHP. When working with NHP, there is always the possibility of anthropo-zoonotic transfer from humans to NHP and vice versa. Moreover, the 3R concept implies that more data are obtained from minimal numbers of animals, and from small samples that are preferably collected using non- or less-invasive sampling techniques. Particularly, New World monkeys (NWM), like marmosets and tamarins, are progressively used in biomedical research. A serious drawback of using NWM is the limited blood sample volume that can be collected. Flow cytometric methods (e.g. Luminex platform), or the analysis of cellular transcripts using microarrays or quantitative RT-PCR assays were developed during the EUPRIM-Net II term to enable researchers to analyse multiple parameters from a single small sample. Here, specific emphasis was laid on the development and application of the above mentioned technologies to monitor biomarkers in NWM and macaques. The implementation of the new technologies in NHP research has significantly contributed to the 3R principle. Use of archival tissue samples, or non-, or less-invasively obtained samples further contributed to the 3Rs, enhancing 1) the quality of life of the animals used in research, and 2) responding to the increasing demand of data by scientists. Importantly, all techniques established in the course of this research project can easily be adapted and applied to other pathogens or diseases of NHP.
Monkey “home office”. Induced stress is a confounding factor for many research questions. Reduction of stress contributes to the refinement of studies by improving the quality of the collected data. As a consequence the number of animals needed for a study is reduced and animal welfare in experimental conditions is improved. An important finding during the EUPRIM-Net II term was that cage-based testing can have an enrichment effect in socially healthy groups in housing colonies of baboons, which may be considered to be the least stress imposing setting investigated here. In a laboratory environment, using cage-based approaches compared to lab-based approaches including animal handling, marmoset monkeys preliminarily show lower acute stress responses. Yet, lab-based approaches with regular handling in marmosets did not lead to sustained increased stress levels. Results were partly obtained from different NHP species and may not transfer to other species. But if cage-based approaches also reduce stress in other NHP species and settings then the cage-based behavioral testing and automated training procedure developed for rhesus monkeys in neuroscientific context could mark a first step towards a refined experimental approach in this field.
Development of in vitro (alternative) technologies. European citizens call for the use of alternative methods replacing animal experiments and testing. In reality, molecular or cell-culture-based methods need validation that is time-consuming. Nevertheless, the development of in vitro methods is a refinement with high potential to reduce the number of animals needed or to even replace an animal experiment and should be pursued. Sometimes an in vitro method can help to alleviate discomfort for the animal undergoing experimental procedures. For these reasons in vitro technologies should be expedited.
Biomedical research that focuses on the central nervous system (CNS) in general goes accompanied with moderate to severe discomfort for animals involved in such research. Still relatively large numbers of NHP are used in in vivo CNS studies, and there is an urgent need to develop alternative in vitro models for NHP CNS studies. The methods developed during the EUPRIM-Net II term are all initiated from ‘left-over’ material from other experiments. We are currently applying these techniques as a pre in vivo test phase, enabling colleagues with valuable information leading to a reduction of animals necessary before the start of in vivo experiments. Not only does this lead to better science with fewer animals, it also makes more complete use of the animals from which the cultures are initiated.
HIV vaccine research requires NHP to be kept under experimental conditions for extended periods of time. The in vitro methods developed within EUPRIM-Net II have the potential to contribute to refinement in HIV vaccine research by reducing the time the animal has to spend in the experimental setting and by helping to distribute animals evenly into the various experimental groups (as means of standardisation) which may even reduce number of experimental animals without losing statistical significance.
Adjuvants are enhancing the immunisation effect but may also cause discomfort to humans but also to research animals in the form of unwanted side-effects. In EUPRIM-Net II in vitro assays were developed to both identify new adjuvant candidates with good adjuvating potential as well as to characterise the granuloma-inducing potential of new adjuvant candidates.
Scientific evaluation of Animal Behavioural Management (ABM) and Positive Reinforcement Training (PRT). Adaptation and training of laboratory primates for participation in procedures are generally accepted as methods to reduce the stress level for the animals and promote more reliable experimental results. However, there is a limited amount of scientific literature within this area and thus little solid information on the effectiveness of measures such as ABM or PRT. EUPRIM-Net II contributed with research in this area and seven articles and a book chapter have been published or are in preparation.
List of Websites:
Contact:
Prof. Stefan Treue (Project Coordinator)
Dr. Björg Pauling (Project Management)
German Primate Center (DPZ)
Kellnerweg 4
37077 Göttingen, Germany
Tel.: +495513851454
FAX: +495513851228
Email: euprim@dpz.eu
URL: www.euprim-net.eu
List of Partners:
Deutsches Primatenzentrum GmbH (DPZ), Göttingen, Germany
Biomedical Primate Research Centre (BPRC), Rijswijk, Netherlands
Department of Health (DH) with
Public Health England (PHE), Porton Down, United Kingdom &
Medicines and Healthcare products Regulatory Agency (MHRA) with
National Institute for Biological Standard and Control (NIBSC), South Mimms, United Kingdom
Westfälische-Wilhelms-Universität (WWU) with
Centre for Reproduction and Andrology (CeRA), Münster, Germany
Consiglio Nazionale delle Ricerche (CNR) with
Institute of Cellular Biology and Neurobiology (IBCN), Rome, Italy
Université de Strasbourg (UdS) with
Centre de Primatologie (CdP), Niederhausbergen, France
Centre National de la Recherche Scientific (CNRS) with
Station de Primatologie (SdP), Rousset, France
Medical Research Council (MRC) with
Centre for Macaques (CFM), Porton Down, United Kingdom
Karolinska Institutet (KI) with
Astrid-Fagraeus Laboratory, Solna, Sweden
The new EU Directive on the protection of animals used for scientific purposes (2010/63/EC) foresees various animal protection and welfare measures. EUPRIM-Net II has contributed ideally to some of them through its different activities. For example Article 28 of the Directive requires the Member States to ensure “that staff be adequately educated, trained and competent”. EUPRIM-Net II regularly offered established courses for scientists, veterinarians, technical and animal care personnel on various topics such as General Primate Biology, Behaviour, Diseases and more. A web-based exchange module for veterinarians and other personnel working with NHPs was developed and implemented. European veterinarians were offered specialised training in NHP requirements in a rotation and training program. Last but not least a NHP-specific laboratory animal science course was developed according to the new (2014) FELASA guidelines. Certification of the course is anticipated before the end of this year (2015). This course is now offered without European funding.
“Psychological wellbeing”, allowing the animals to express natural behaviours and gain a measure of control over their environment, is addressed through the field of Animal Behavioural Management (ABM) as well as Positive Reinforcement Training (PRT). Training is now offered in eight different lectures by the EUPRIM-Net ABM/PRT seminar group. What makes this concept unique is that the lectures have been standardised and translated into six European languages meeting the demand for training by local trainers in the mother tongue. The seminar group was supported by research activities to scientifically evaluate the effectiveness of ABM/PRT through behavioural observations by trained ethologists and the assessment of physiological effects.
Knowledge exchange between experts is the basis for advancement in science. EUPRIM-Net II organised a series of workshops and conferences on relevant topics around primate research and keeping, this included events especially targeting the industry as well as non-European institutes in order to maximise outreach.
Although for the time being the total replacement of NHPs in vivo experiments by in vitro models is far from feasible, ethical considerations compel the development of alternative methods. Consequently, with Article 10 the Directive 2010/63/EC “seeks to facilitate and promote the advancement of alternative approaches”, and (the validation of) alternative methods are subject of Articles 46 and 47. EUPRIM-Net II has developed in vitro technologies to replace, reduce and refine NHPs studies within neuroscience/neuroimmunology and vaccine research.
Biobanking is another approach to reduce the number of animals in experiments. Article 27 of Directive 2010/63/EC states that “Member States should, where appropriate, facilitate the establishment of programmes for sharing the organs and tissue of animals that are killed”. The EUPRIM-Net BioBank has provided access to primate materials from different NHP species.
The research projects also developed and improved assays for the health monitoring of NHP. As important refinement cage-based testing systems were developed and advanced, reducing the stress of animals in experiments.
Project Context and Objectives:
The development of alternative technologies promises to replace testing on animals. However, for the foreseeable future until valid alternatives exist, biological and biomedical research as well as toxicological safety testing depends on the use of relevant animal models. In some cases the animal model of choice are non-human primates (NHP). Although this group forms less than 0.1% of all animals used in research, NHP play a special role in providing critical insights that are central for many areas of research owing to their highly developed sensory and cognitive abilities. At the same time research with NHP underlies particularly high ethical (and legal) standards, and a responsible application of the 3Rs concept of Refinement, Reduction, and Replacement is mandatory.
The EU and its national states have instituted a large number of measures to ensure the best welfare of NHP used for scientific purposes. Besides legislative and regulatory efforts (Directive 2010/63/EC on the protection of animals used for scientific purposes) the EU has provided funding to EUPRIM-Net, a network of nine European primate centres from six countries which was established as a Research Infrastructure in 2006 and has now just completed its second round of funding by the Commission (FP7-GA-262443).
In this project the primate centres’ infrastructures and expertise were integrated in order to provide critical services, training and advice to scientific institutions in Europe conducting primate research and to zoological gardens that keep primates. The Network-, Access- and Research Activities all aimed at advancing animal welfare and the 3Rs for NHP in experiments and procedures.
Best Practice in Husbandry and Experimental Procedures. Knowledge exchange and networking is probably one of the most important activities for researchers in order to advance their science and progress. What better occasions can yield opportunities than workshops and conferences on topics in their field? EUPRIM-Net’s aim was to provide a series of workshops on relevant topics all around primate keeping, handling and research. The target audience were primate veterinarians and scientists working in primate research, including the industry and non-European primate centres as important partners and stakeholders.
Veterinarians specialised on non-human primates are rare and require a specialised education before being able to assess the special requirements and needs of NHP. EUPRIM-Net II aimed at contributing to the education of veterinarians in becoming NHP-specialists by giving European veterinarians the opportunity to work hands-on with monkeys. Implementation of an electronic exchange module was aimed to facilitate communication between veterinarians from different primate centres.
ABM/PRT. Behavioural Management techniques and Positive Reinforcement Training (PRT) of laboratory NHPs for participation in procedures has been developed and implemented within EUPRIM-Net to minimise the stress level for the animals, promote more reliable experimental results, and lead to an increased safety, both for animals and personnel. “Psychological wellbeing”, allowing the animals to express natural behaviours and gain a measure of control over their environment is a core aim within EUPRIM-Net. Psychological wellbeing is addressed through the field of Animal Behavioural Management (ABM), involving ethological aspects of captive animal husbandry as well as PRT. In EUPRIM-Net II the aim was to establish an ABM/PRT seminar group in order to spread knowledge and understanding about ABM and PRT of laboratory primates to as many primate facilities as possible in Europe and around the world. The seminar group should be supported by research activities to scientifically evaluate the effectiveness of ABM/PRT through behavioural observations by trained ethologists and the assessment of physiological effects.
Education. Article 28 of the Directive requires the Member States to ensure “that staff be adequately educated, trained and competent”. Therefore, education and training of staff working hands-on with NHP was one of the most important aims of EUPRIM-Net II. For veterinarians and scientists courses were planned on different subjects (modules): General Biology, Behaviour, Husbandry, Diseases and Parasites, Ethics; animal caretakers and technicians have a more difficult time than scientists to find suitable CPD courses. EUPRIM-Net II planned to provide an opportunity for animal caretakers and technicians in primate research to continue their education and improve their skills. For animal caretakers and technicians it may be necessary to provide courses in their mother tongue as they may not be as proficient in English as scientists. To guarantee standardisation and high quality of the courses, certification of the courses by the Federation of European Laboratory Animal Science Associations (FELASA) was aspired.
International Cooperation. In order to maximise outreach EUPRIM-Net aspired to establish a comprehensive exchange programme between important international key players in primate research. EUPRIM-Net aimed to offer a platform of international collaborations linking the centres’ expertise in all relevant areas and resulting in manifold benefits for all participating centres, including EUPRIM-Net.
Co-operation with industrial/commercial partners. In addition to primate centres functioning as public bodies several commercial companies use primates to an extensive degree and thus should be involved in the ongoing discussion and development of the use of primate for research and other purposes. One of EUPRIM-Net II’s objectives was to intensify the collaboration with these industrial partners to exchange and learn from the industry’s experience as well as let them participate in the activities of EUPRIM-Net.
PRIMOCID. With PRIMOCID the objective was to provide access to well-established disease models with the aim to facilitate studies on disease mechanisms and the development of new therapies. The aim was to offer Access to models for rheumatoid arthritis (in rhesus monkeys and marmosets), multiple sclerosis (in rhesus monkeys and marmosets), allograft rejection (skin, in rhesus monkeys), and (new to this project) to infectious disease models and neurodegenerative diseases. Projects aimed at the development of alternatives for animal testing, such as organotypic cultures or refinement of existing models was appreciated. Development of new disease models was also possible, but on a limited scale as this should fit within the limitations for access provided with regard to time and budget. In addition to the disease models access was to be given to the primate laboratories at the BPRC and the possibility to study primate behaviour.
BioBank. Tissue and genetic material obtained from primates represents a valuable resource as it can be used in specific studies in biomedical research. However, the provision of primate material is difficult and time demanding and hence, comparative studies involving primate research are often challenging or even impeded due to limited or non-availability of appropriate material. The aim of EUPRIM-Net II was to make available collections of diverse primate materials in three institutes of EUPRIM-Net (DPZ, BPRC, WWU) both for internal use and for external scientists. The idea behind the BioBank is to ensure the sharing of rare and valuable primate resources through more efficient access to specific types of samples available to researchers for biomedical research as well as for conservation studies. The EUPRIM-Net II BioBank aimed to: 1) accelerate progress in the solution of biomedical and other scientific problems that depend on supply of primate biological materials; 2) contribute to the 3R-concept (reduce, refine, replace) by reducing the numbers of primates needed through efficient resource sharing; 3) substantially enhance various European research fields through access to restricted primate materials, such as non-invasively collected DNA samples from endangered species, RNA and cDNA samples from a variety of healthy and diseases tissues; 4) provide genetically characterized and, therefore, certified primate material. The EUPRIM-Net II BioBank consists of: A) a tissue bank representing both normal and diseased tissues of primates including blood and serum samples (DPZ, BPRC, WWU); and B) a gene bank with DNA, RNA, cDNA.
Tools to Measure Infection and Diseases. Further EUPRIM-Net aimed to develop and apply new technologies for the detection and validation of diagnostic and prognostic markers of viral and bacterial diseases in NHP. The specific objectives were: 1) to develop multiplex assays for detection of pathogens, and for the determination of immune responses and cytokine levels; 2) to develop transcript-analysis assays for the identification of diagnostic and prognostic RNA markers of disease progression; 3) to explore the use of non-invasive, or less-invasive sampling in these assays, 4) to standardise protocols and exchange data between partners.
The implementation of the new technologies in NHP research has a high merit to strongly contribute to the 3R principle. Use of archival tissue samples, or non-, or less-invasively obtained samples further contributes to the 3R’s. Importantly, all techniques established in course of this research project can easily be adapted and applied to other pathogens or diseases of NHP. The techniques developed in this Research Activity will have considerable impact on future research in the EU involving NHP. This will not be limited to the field of infectious diseases, as they can be widely used in research on animal models for human diseases.
Cage-based Systems. Recording of physiological data, especially invasively, but also non-invasively, often requires that animals are handled and restrained in their movements during the experimental procedures. In system neuroscience, for example, constraining the animal’s head movements is the standard procedure for microelectrode recordings from the animal’s brain, while the animal performs cognitive tasks. The interaction with the animal bears the potential to induce stress, which should be avoided, might be detrimental to the behavioural learning progresses, and can have confounding effects on the physiological parameters in question. If physiological data is not collected for research purposes, but to monitor animal welfare in a colony, such stress leads to the paradoxical situation that attempts to optimize animal welfare might actually reduce it. The objective of this research activity was to develop and implement procedures for remotely monitoring physiological and behavioural data from monkeys in their housing environments. Telemetry systems were combined with behavioural test systems for integrated and automated data collection. The expected impact is a considerable reduction of potential stress for monkeys in experimental situations, and improved possibilities to continuously monitor animal welfare. These developments help to identify sources of stress and to reduce stress in research monkeys. Induced stress is a confounding factor for many research questions. Reduction of stress contributes to the refinement of studies by improving the quality of the collected data. As a consequence the number of animals needed for a study is reduced and animal welfare in experimental conditions is improved.
In vitro Technologies (Alternative Methods). Another important aim of this project was to develop and apply new in vitro technologies leading to Replacement, Reduction and Refinement (the 3Rs) regarding the use of NHP in biomedical research. The specific objectives were: 1) to develop in vitro model systems for research on the central nervous system; 2) to develop in vitro test methods for vaccine research.
Studies on the CNS that use NHP often go accompanied with moderate to severe discomfort for the animals. This is often due to the invasive nature of the studies or to the very long preclinical incubation times of e.g. transmissible neurodegenerative diseases. Moreover, the negative public perception of CNS studies provides an extra motivation for the development of alternatives. The development and validation of in vitro primary cell cultures, transgenic cells and specified cell culture systems can provide researchers with the possibility to predict the outcome of in vivo experiments and to screen compounds or therapeutics that directly target resident CNS cells as a pre-in vivo test phase, reducing the number of NHP needed for in vivo studies. The development and validation of slice culture techniques can be used to address even more complex research questions, further reducing animal numbers for in vivo studies.
In HIV vaccine research, controlled prime-boost regimen strategies followed by experimental exposure (challenge) to simian or human/simian immunodeficiency viruses (SIV or SHIV) is, as yet, only feasible in NHP. The protective outcome of SIV vaccination can only be measured in vivo, leading to studies where NHP are kept under experimental conditions for extended periods of time. The aim was to develop and validate in vitro methods to predict the clinical outcome of vaccination ex vivo, ideally rendering the experimental in vivo challenge with virus unnecessary leading to a considerable reduction of the duration of in vivo experiments. One of the major obstacles in the development of vaccines is the selection of potent adjuvants that are safe and licensed for human application. Adjuvants and vaccine formulations are tested in various animal models, including NHP, causing significant degrees of discomfort in the form of unwanted side-effects. The objective was to develop and employ in vitro technology to select and develop new adjuvants with good adjuvating potential, but with minimal side-effects. Although this project contributes mainly to refinement, the topic is so general and involves so many animals that even slight improvements will generate considerable effects regarding animal welfare.
Project Results:
Best practice and harmonisation in husbandry and experimental procedures with NHP. Knowledge exchange and networking is probably one of the most important activities for researchers in order to advance their science and progress. To this end, twelve workshops were organised targeting veterinarians and scientists and one also addressing regulatory authorities as well as the public. Connections to other European organisations focusing on non-human primates [European Primate Veterinarians (EPV)] and professional animal training experts [AnimalConcepts] were exploited. The NHP-using industry and non-European primate centres are valuable exchange- and collaborative partners, thus five workshops were especially targeted towards these groups. One workshop in Nairobi, Kenya included a minisymposium that was attended by the German Ambassador in Kenya. All in all the workshops were visited by more than 400 participants.
EUPRIM-Net II workshops between 2011 and 2015 – brief summaries:
“General techniques and best practice using NHP”: This workshop was co-hosted by EPV and focused on minimal invasive techniques to be used in both research centres and zoos. Besides presentations and discussions, training on endoscopic techniques by the experts in a “dummy system” and more detailed information on thermographic techniques were provided.
“Primate Welfare & Training Workshop”: A summary of the workshop has been published in “Enrichment Record” with both Euprim-Net members (Marit Vernes) and AnimalConcepts (Sabrina Brando) as co-authors. [Brando S, Vernes M (2014). Primate Welfare and Training Workshop. Enrichment Records, Winter 2014]
“Food-and-fluid control”: The workshop was co-hosted by AnimalConcepts; the aim was to share knowledge on training methods to refine the use and care of NHP in neuroscience research. Veterinary care of these animals, the possibilities to prevent or diminish food and fluid control and behavioral management were discussed.
“Primate nutrition and its implications in research and on animal health”: The workshop was co-hosted by EPV and included effects of primate nutrition on various aspects of animal welfare and behavioral management. In addition, several case reports discussing practical aspects were presented and discussed. These discussions included topics on food supplements, food restriction, obesity, and skin problems.
“Workshop on the microbiome and animal health”: A one-day workshop on the microbiome and animal health, specifically aimed at non-human primates. The discussions included practical aspects on analysis and more detailed effects of changes in the microbiome and health aspects.
“Non-invasive imaging in non-human primates”: A workshop on non-invasive imaging in non-human primates was conducted including Refinement of infectious disease studies using advanced imaging; SPECT in non‐human primates; Optical imaging in preclinical research; Hyperpolarized MRI – Potential and perspectives; Structural and functional cardiac imaging; Research in non‐human primates – role of in vivo imaging; Anatomical and functional MRI in awake marmosets; Diffusion weighted imaging: Basic concepts and recent technological advances; Arterial spin labeling: Non‐invasive perfusion measurements and beyond; Neurotransmission and brain metabolism - Advanced magnetic resonance spectroscopy.
“Methods and Practices for professional banking of tissues and material”: One of the main issues was the visibility and how to reach the general researchers. From this point of view, much input was provided by the users and the external speakers.
“Alternative methods for the use of non-human primates in biomedical research”: The workshop included lectures by key scientists in the field of alternatives as well as by experts from governmental and non-governmental organizations. Furthermore, parallel sessions were organised to stimulate discussion on the challenges of advancing the use of alternative methods for NHP. Subgroups voted on four statements and together composed a list with opportunities and priorities. [ALTEX. 2014;31(4):520-9. doi: http://dx.doi.org/10.14573/altex.1406231(se abrirá en una nueva ventana). Epub 2014 Jul 24.]
Workshops conducted together with industrial and/or non-European partners:
“Positive Reinforcement Training (PRT) and Animal Welfare“ and “Practical training and implementation of environmental enrichment”: These two workshops were conducted at the Institute of Primate Research (IPR) in Nairobi, Kenya. The Basics of PRT were taught and the possibilities of implementing enrichment practically demonstrated. One of the workshops was accompanied by a minisymposium attended by the German Ambassador.
“Novel Insights into Primate Genomics: Impact for Biomedical Research“: This workshop was initiated by our industrial partner and co-hosted by EUPRIM-Net because species selection and suitability of an individual experimental animal may depend on the genetic background, which may not always be known. [Trends Genet. 2014 Nov;30(11):482-7. doi: 10.1016/j.tig.2014.05.004]
“Refinement of non‐human primate use in research“: This workshop was initiated by our industrial partner as satellite workshop at the symposium of the International Primatological Society and co-hosted by EUPRIM-Net because an important part of this workshop was on education of staff working with NHP.
“Workshop on the use and the advantages of marmosets in translational biomedical research”: Genetically modified marmosets most likely will become exceptionally relevant in academic and industrial research in Europe, the US, and Japan. However, since important biological characteristics of this NHP species may be affected in genetically modified marmosets, it is of utmost importance to proactively consider possible effects of the modifications on the monkeys’ well-being and housing. In this context, the 3R (Reduction, Refinement, Replacement) and 4R (3Rs plus) principles were discussed.
Education & Training. Article 28 of the Directive requires the Member States to ensure “that staff be adequately educated, trained and competent”. To this end one of the most important aims of EUPRIM-Net II was to provide education to staff working hands-on with primates, including all occupations involved from animal care takers, animal trainers and technicians to scientists and veterinarians. Participants came from ten different European countries and four non-European countries.
Animal care takers, animal trainers, technicians:
This group has a more difficult time than scientists to find suitable Continued Professional Development (CPD) courses. EUPRIM-Net II provided a highly appreciated opportunity for animal caretakers and technicians in primate research to continue their education and improve their skills. For them it may be necessary to provide courses in their mother tongue as they may not be as proficient in English as scientists. This was taken into consideration. More than 125 participants took part in six courses carried out in Germany and the UK. Content of the courses were general primate biology, diseases and parasites, husbandry, handling, and its challenges (e.g. zoonses), communication of primate research (problems, strategies and suggestions).
Veterinarians:
The rotation system for training of veterinarians and assistants/technicians was planned to foster species-specialisation of veterinarians with regard to non-human primates. The programme admitted six veterinarians who were trained in two-week training visits at the BPRC visits. After each training period, participants received a training certificate.
Veterinarians and scientists:
Ten CPD courses were offered to veterinarians and scientists that were in total attended by 210 participants. Course contents were offered in modules on general biology; primate sociobiology, husbandry, and nutrition; primates in biomedical research; primate husbandry, medical aspects, security, advanced methods and ethics; general primatology, behaviour, cognition and neurophysiology; marmosets as animal models. The course language was English or French.
In the beginning of 2014 FELASA (Federation of European Laboratory Animal Science Associations) revised their recommendations for the accreditation of education and training courses in laboratory animal science. According to the new system it became possible to introduce species-specific courses that could be certified by FELASA. This opened up the way for EUPRIM-Net II to develop primate–specific Laboratory Animal Science (LAS) courses according to FELASA guidelines, function A+B, the highest recognised certification in Europe and address Continued Professional Development as mentioned in Annex I. The first course was held within the term of EUPRIM-Net II. This course was divided in two parts, encompassing an e-learning phase that was specifically developed for this course as well as practical parts. The accreditation process of the primate-specific Laboratory Animal Science course is ongoing beyond the term of EUPRIM-Net II. Nevertheless, development of this course would not have been possible without the support of the EU Commission.
All staff working hands-on with monkeys:
A Seminar Group has been formed, consisting of veterinarians, ethologists, and animal trainers. Together, this group has created eight lectures on topics regarding Animal Behaviour Management (ABM), ranging from breeding laboratory primates to enrichment and problem solving. The group now offers to come to facilities keeping NHP and present these lectures as in-house training. They can customise a combination of lectures according to the needs of the facility; for instance, the facility might be interested in learning more about how to conduct behavioural studies and how to start training their primates. All lectures are 3-4 hours long and the target audience is animal technicians; however all personnel involved in managing the colony, including scientists, will benefit from attending. During the EUPRIM-Net II term (2011-2015) 19 seminars with one or more lectures were given to NHP-keeping facilities within and outside Europe of which only a basic number was subsidised by the EU; since then the in-house trainings have become well-known and popular and requests have already come in for the time beyond EU-funding for EUPRIM-Net II. A short description of the different lectures can be found below.
Lectures offered by the ABM/PRT Seminar Group (www.euprim-net.eu/network/ABM-seminars.html):
Ecology and behaviour of primates - In this lecture natural including social behaviour of common laboratory primates is described - important knowledge when housing a particular species.
How to optimize laboratory primate housing and cages - An empty cage is useless to the animals. This lecture gives ideas of how to plan and improve the animals' living quarters. Improved welfare may improve the quality of the science!
An introduction to primate training - This lecture gives the basic tools to start training animals to collaborate in procedures, facilitate housing and handling, and improve human-animal relationships!
Environmental enrichment in primate facilities - Why and how a modern enrichment program may be implemented is presented in this lecture in a structured approach.
Breeding laboratory primates - How can breeding success be optimised? This lecture gives ideas with regards to primate breeding, incorporating veterinary, behavioural and practical perspectives.
Solving behavioural problems - Animals don't always behave the way we want them to. In this lecture, structured approach is presented to solving common behavioural problems in primate facilities.
How to observe and measure behaviour - Did the enrichment programme work? Which individuals are compatible for pair housing? From a management perspective observing and measuring behaviour is capital. This lecture adresses those issues.
Why stress matters - In this lecture, we argue that we need to be concerned with reducing stress both to improve welfare and the quality of the science.
Outreach & Dissemination. The EUPRIM‐Net consortium takes serious interest in providing the public community with solid information about the keeping and wellbeing NHP used for research purposes in the primate centres of the Infrastructure and their benefit for health research. This is especially important in the current setting, where recently various ways (European citizens’ initiative, European Parliament resolutions, petitions, and parliamentary questions) have been attempted to call into question the value of Directive 2010/63/EU, to try and repeal it and ban the use of animals in research and development.
To this end factual reporting and information sharing on research with and on NHP is more crucial than ever. Therefore, a virtual tour was designed and developed that was regarded an appropriate approach to inform a broad audience about the breeding and housing facilities and the way non‐human primates are bred, kept and handled for research purposes in primate centres. For the virtual tour (www.dpz.eu/en/info‐center/mediacenter/virtual‐tour.html) the premises of the German Primate Center (DPZ) were chosen as an applicable example also reflecting the work of the other EUPRIM‐Net institutions.
BioBanking. Over the recent years the value of biobanking and its contribution to the 3Rs (replacement, reduction, refinement) has become more and more obvious. This has been recognised by European legislation and put in writing with Directive 2010/63/EU on the protection of animals used for scientific purposes: Article 18 urges member states to facilitate where appropriate, the establishment of programmes for the sharing of organs and tissues of animals killed. And in fact one such programme has been established by the EU‐funded infrastructure EUPRIM‐Net with the EUPRIM‐Net BioBank.
Over its 54 months duration EUPRIM‐Net II has provided 97 organ‐ and tissue samples of non‐human primates to 21 research projects. Since maximally 20% of the total infrastructure can be subsidised by the EU that means that at least 485 organ‐ and tissue‐samples have been distributed by the EUPRIM‐Net institutes involved. It is actually more likely that all in all close to 600 samples have been provided by the consortium over the EUPRIM‐Net II duration, as one institute was not involved in the EUPRIM‐Net BioBank at the time of proposal submission but since then have established a biobank and have distributed non‐human primate samples. One institute was involved but did in fact not distribute samples via the EUPRIM‐Net biobank due to the complexity of the administration.
The number of samples distributed via the EUPRIM‐Net BioBank reflects the ratio of non‐human primates versus other species, especially rodents, used for research purposes. Considering this niche the number of distributed samples is actually quite impressing, especially with respect to the 3Rs and the number of animals which may have been spared. The research projects that were provided with organ‐ and tissue samples were in the fields of immunology, oncology, endocrinology, neuro‐ and eye anatomy, neurophysiology, drug screening, genetics and epigenetics, and HIV research.
Gene samples from non‐human primates are rare and yet a variety of scientific disciplines can make good use of them. Whether used in research on conservation issues, evolution and epigenomics, genetic backgrounds of social behaviours or for conducting medical research like finding cures for diseases like B‐cell‐malignancies or HIV: DNA or RNA of different NHP species, from Rhesus or Cynomolgus macaque to chimpanzee and bonobo from the EUPRIM-Net II BioBank have contributed to scientific progress during the past 54 months and were much appreciated by the scientists.
All in all, the EUPRIM‐Net Genebanks provided 80 samples that were funded by the European Commission via EUPRIM‐Net II to 17 research projects. As maximally 20% of the total infrastructure can be reimbursed by the EU that means that over the 54 months of EUPRIM‐Net II funding at least 400 gene samples were provided to the European scientific community.
PRIMOCID. With PRIMOCID (Non-Human Primate Models of Chronic Immune Disorders) Transnational Access was provided to eight research projects, supporting scientists from six different European countries with experimental animals and expertise. The projects were in the fields of Multiple Sclerosis, Tuberculosis, Malaria, Alzheimer Disease, DNA vaccines, arthritis, and behaviour.
Tools to measure Infection and Disease in NHP. The 3R concept implies that more data are obtained from minimal numbers of animals, and from small samples that are preferably collected using non- or less-invasive sampling techniques. To this end several assays have been developed in EUPRIM-Net II:
When working with NHP, there is always the possibility of anthropo-zoonotic transfer from humans to NHP and vice versa. To better assess the risk of infection a multiplex assay for the detection of respiratory pathogens in NHP was developed and validated. Using this assay, infections with respiratory viruses could be detected in colonies of NHP. For the direct detection of different viruses, a Luminex-based multiplex assay, the so called Respiratory Luminex assay (RLA), was developed and validated. The same technology platform has been used to identify cytokine signatures in materials from macaques infected with Mycobacterium tuberculosis (Mtb) and evaluate their potential to provide diagnostic indicators.
Characterisation of the gut microbiome from the colony of healthy rhesus macaques at the BPRC was started and feces of animals of different age groups and sex were sampled. Libraries of 16S-rRNA were prepared and further sequenced. At least three new bacterial species were identified and have been or will be submitted to the German Collection of Microorganisms and Cell Culture (Leibniz-Institute DSMZ) for further in-depth characterisation and inclusion in their collection. Currently, a new Peptococcus strain has been identified in the rhesus macaques (proposed name: Peptococcus simiae), and has been submitted to this collection. Several other new bacterial strains will also be sent out to the DSMZ for in depth analysis soon. This work will be continued to better understand the reciprocity of the immune system with the microbial community of the gut.
Alternative approaches to dissecting immune responses in GBV-B-infected tamarins were explored (GBV-B is the tamarin counterpart of the hepatitis C virus). The GBV-B detection system was improved by using an extensive archive of a range of biological materials from naive and infected animals as well as new animal studies where appropriate.
More assays were developed for the identification of diagnostic and prognostic markers of viral and bacterial diseases in NHP. These apply transcript analysis and have a potential to improve health surveillance programs in NHP-keeping facilities. E.g. MPXV-regulated miRNAs could be detected in samples from infected macaques and cell cultures. Moreover, Herpes B Latency associated transcript (LAT) could be detected and eight new miRNAs identified in herpes B virus (BV)-infected cells. Complementing this assay, a quantitative Polymerase Chain Reaction (qPCR) assay for the detection of BV-specific nucleic acids in ganglia of rhesus macaques could be established. A peptide microarray technique was used to develop an assay to measure antibodies against the two immunodominant glycoproteins B (gB) and D (gD). This should avoid “false negative” results possibly obtained by the commercially available HSV-1-ELISA.
Dedicated mRNA signatures from Mtb-infected macaques could be validated that are prognostic RNA markers of disease progression and miRNAs associated with the pathology of viral hepatitis were explored as a result of GBV-B infection in tamarins.
Non-invasive or less-invasive sampling for use in multiplex or transcript-based assays was explored and the development of non-invasive sampling procedures for routine virus surveillance was initiated. This work has not been completed; nevertheless, in total more than 400 swabs have been collected from the BPRC colony animals and stored at -80°C.
Proof of concept was established for the use of dried blood spots for evaluation of cytokines using a Luminex multiplex immunoassay. Exploring the use of non-invasively sampled materials for the detection of miRNA has started. Literature suggests that miRNA can be detected in urine, and thus urine samples taken at termination have been stored in order to perform comparisons with assays undertaken on serum and liver when these are fully analysed.
Cage-based wireless recordings in unrestrained monkeys. Recording of physiological data, especially invasively, but also non-invasively, often requires that animals are handled and restrained in their movements during the experimental procedures. The interaction with the animal bears the potential to induce stress, which should be avoided, might be detrimental to the behavioural learning progresses, and can have confounding effects on the physiological parameters in question. If physiological data is not collected for research purposes, but to monitor animal welfare in a colony, such stress leads to the paradoxical situation that attempts to optimise animal welfare might actually reduce it. The research reported here had two major goals. First, comparison of stress levels in different experimental procedures in primates, particularly testing for the effect of a cage-based behavioral testing approaches compared to approaches which require handling the animals; second, development of a cage-based behavioral testing and training procedures for rhesus monkeys suitable for neuroscientific research. The major finding of this reporting period and WP9 as a whole supports a cascade of stress level increments one may have expected: Cage-based testing can have an enrichment effect in socially healthy groups in housing colonies of baboons, which may be considered to be the least stress imposing setting investigated here. How a cage-based test system in a lab environment and in different species compares to that remains to be investigated. In a laboratory environment, using cage-based approaches compared to lab-based approaches including animal handling, in marmoset monkeys preliminarily show lower acute stress responses in cage-based approaches compared to lab-based approaches. Yet, lab-based approaches with regular handling in marmosets did not lead to sustained increased stress levels which would show in sleep or other physiological stress-associated parameters measured remotely. Results were partly obtained from different NHP species and may not transfer to other species. But if cage-based approaches also reduce stress in other NHP species and settings then the cage-based behavioral testing and automated training procedure developed for rhesus monkeys in neuroscientific context in EUPRIM-Net II could mark a first step towards a refined experimental approach in this field.
In vitro technologies to replace, reduce, and refine NHP studies. For the time being biological and biomedical research as well as toxicological safety testing depends on the use of relevant animal models. In some cases the animal model of choice are NHP. Nevertheless, the number of animals used in experiments ought to be as small as possible. To this end EUPRIM-Net II has conducted research to develop in vivo technologies that can deliver (preliminary) results necessitating fewer NHP in the experiment.
This research was able to validate primary in vitro glia cell cultures as pre in vivo screening method. Some tissue-specific findings in this research project might be therapeutically exploitable in neuro-inflammatory and neurodegenerative diseases. Furthermore organotypic brain slice cultures from rhesus macaques were characterised: Although these slices were demonstrated to be responsive to neuro-inflammatory stimuli, exposure to neurotoxic agents resulted in massive cell death and general loss of viability. Whereas the organotypic slice cultures thus represent a valuable new tool to study (innate) neuro-inflammatory responses, their use as models for neurotoxicity is limited.
For the study of BSE and transmissible neurodegenerative diseases in NHP in general, there is a lack of an appropriate in vitro system. The strategy was the establishment of transgenic rabbit kidney (RK13) cell lines which overexpress full-length prion protein. Established transgenic cell lines expressing PrPc of either squirrel monkey (Saimiri sciureus) or cynomolgus monkey (Macaca fascicularis) BSE did support prion propagation and therefore could not be used for the diagnosis of macaque-adapted BSE; however RT-QuIC could be established and adapted for the ultrasensitive detection of prion activity in various specimens. With this new technique prion replication in peripheral tissues of BSE-inoculated macaques can be assessed. The detection of prions in different clinical specimens (e.g. CSF, lymph nodes, blood), that can be obtained routinely and/or by mildly invasive methods and even before the occurrence of clinical manifestations is of high importance. Those clinical specimens in combination with ultrasensitive methods, like RT-QuIC, can be used to predict disease outcome in vivo thereby significantly reducing the time necessary for the keeping of prion-inoculated macaques. We therefore consider the establishment of RT-QuIC for specific and highly sensitive detection of macaque-adapted BSE as equivalent to the anticipated deliverable to produce BSE-susceptible cell lines.
Although infection rates are declining due to improved education and enhanced worldwide access to antiretroviral medication, HIV-infection remains a global health problem. However, an AIDS vaccine is still lacking and NHP research remains a necessity to reach that goal. An in vitro test that delivers early prediction of AIDS vaccine efficacy in human or macaques, which represent the major species for preclinical AIDS vaccine trials, could contribute to vaccine development as well as to refinement and reduction of studies conducted in NHP. We therefore aimed at developing predictive in vitro protocols in the monkey model of AIDS. To do so, we primarily analysed samples from three different experiments. Extending the original application, we used four approaches to refine the macaque model of AIDS: 1) We validated and/or detected other prognostic marker for the macaque model of AIDS; 2) As a potential additional prognostic marker, we quantified expression of innate immunity genes and identified correlates of protection that can be used to determine AIDS vaccine efficacy ex vivo within 48 h post vaccination. 3) We found that a single nucleotide polymorphism (SNP) in DC-SIGN/CD209 - that is similar to a human SNP - can be used as a further prognostic marker to distribute animals evenly into the various experimental groups (as means of standardization) to reduce number of experimental animals without losing statistical significance (manuscript in preparation). 4) We investigated antiviral non-cytolytic activity of CD8 cells in an attempt to identify a novel prognostic marker that can serve as standardization of experiments. While we did not find strong evidence that this activity influenced susceptibility to SIV-infection, candidate genes linked to this activity were detected by transcriptional profiling.
Adjuvants are formulations, which upon administration lead to non-specific immune stimulation. They are used to induce immune responses directed against pathogens (as for vaccination purposes) or to generate immune responses against components of the body itself (as in experimental animal models of human auto-immune diseases like e.g. multiple sclerosis, diabetes or rheumatoid arthritis). However, some of the more potent adjuvants are notorious for their adverse effects. Complete Freund’s Adjuvant (CFA) for example is known for the development of granulomatous skin lesions, hampering application in humans and causing various degrees of discomfort to NHP. There is therefore an urgent need for new and safe adjuvants for human use and a particular need to develop new, cleaner adjuvants for use in experimental animals. With the recent discovery of a family of molecules that form part of the innate immune system, the Toll-like and NOD-like receptors (TLR and NLR resp.), important progress was made in our understanding of how adjuvants work on a molecular level. Upon activation these receptors deliver a potent non-specific activating signal to the immune system, much like adjuvants do. We have used this knowledge to develop in vitro systems to model the adjuvating effects, in the form of TLR and NLR bioassays, as well as in vitro systems to model the adverse effects, in the form of in vitro granuloma models. Both assay types have been developed and extensively used to both identify new adjuvant candidates with good adjuvating potential as well as to characterise the granuloma-inducing potential of new adjuvant candidates.
Moreover alternative approaches to CFA have been developed using mycobacterial strains that specifically lack the expression of sets of proteins inducing less lung granulomas than CFA. Second, as a bottom-up approach, we have characterised the overall innate immune responses evoked by M. butyricum, both qualitatively and quantitatively, and used this information to compose an entirely new adjuvant (MiMyc) that mimics these responses.
Scientific evaluation of Animal Behavioural Management (ABM) and Positive Reinforcement Training (PRT). Research was carried out within EUPRIM-Net II to investigate effects of ABM/PRT measures or experimental refinement methods. In this context the impact of voluntary cognitive testing on captive group-housed long-tailed macaques’ (Macaca fascicularis) stress level and social behavior was examined. There are two opposing views on the potential impact of experimental testing: Separation from the group and/or frustration during the experiment might cause an increased stress level. Alternatively, experimental testing might serve as enrichment for captive individuals and thus contributes to the animals’ welfare. When data collection will be finished (September 2015), we will pool the data to enhance sample size for the planned paper “The impact of cognitive testing on stress levels in captive long-tailed macaques (Macaca fascicularis)”. Overall, the results strongly suggest that cognitive testing is neither detrimental to the participating individuals nor to the group as a whole, in terms of agonistic interaction rates.
Moreover decision making in animal trainers was analysed as a function of training frequency and a paper finalised that has recently been submitted for publication. A textbook chapter was written on laboratory primate training that will shortly be submitted for publication in an online, open access format. During EUPRIM-Net II at least seven papers and five additional manuscripts in the field of ABM/PRT were produced in different stages of publication. Note that two papers also explore intricate uses of negative reinforcement training (NRT) in combination with PRT. An interesting finding was that NRT may indeed be used to reduce fear and give animals more control over their situation. Data collection has started on the impact of early socialization on laboratory primates. This project has stalled but we’re hoping to relaunch it during 2016. Blood cortisol data was analysed from animals in different stages of training. However, the results were inconclusive and we’ve decided not to pursue a publication elaborating on that issue.
Potential Impact:
For the foreseeable future until valid alternatives exist, biological and biomedical research as well as toxicological safety testing depends on the use of relevant animal models. In some cases the animal model of choice are non-human primates (NHP). Understanding the biology of nonhuman primates helps us to understand our own biology. That is why NHP play an important role in biological and biomedical research. This group of animals provides models which are essential for research in genomics and biotechnology for health. The successful development of new strategies against human diseases like infectious (e.g. HIV, hepatitis) and neurological diseases (e.g. Alzheimer´s or Parkinson Disease) or cancer depends on the availability of NHP or biological material of NHP origin. This also holds true for the development of new therapeutics (vaccines, gene therapy) and transplantation research. Although this group forms less than 0.1% of all animals used in research, NHP play a special role in providing critical insights that are central for many areas of research owing to their highly developed sensory and cognitive abilities. At the same time and for the same reasons research with NHP underlies particularly high ethical (and legal) standards, and a responsible application of the 3Rs concept of Refinement, Reduction, and Replacement is mandatory.
This is especially important in the current setting, where recently various ways (European citizens’ initiative, European Parliament resolutions, petitions, and parliamentary questions) have been attempted to call into question the value of Directive 2010/63/EU on the protection of animals used for scientific purposes, to try and repeal it and ban the use of animals in research and development. To this end factual reporting and information sharing on research with and on NHP is more crucial than ever.
The EU and its national states have instituted a large number of measures to ensure the best welfare of NHP used for scientific purposes. Besides legislative and regulatory efforts (Directive 2010/63/EU) the EU has provided funding to EUPRIM-Net, a network of nine European primate centres from six countries which was established as a Research Infrastructure in 2006 and has now just completed its second round of funding by the Commission (FP7-GA-262443).
Outreach. With EUPRIM-Net II the EU has continued support to a virtual European Primate Centre where standards are high, best practices are used and all NHP-keeping facilities in Europe and worldwide can benefit. Owing to their highly developed sensory and cognitive abilities, it is important to exchange and network for the welfare of the NHP in experiments. If the experimental animal is stressed this may have confounding effects to the physiological parameters in question and thus detrimental to the advancement of biological and biomedical research. EUPRIM-Net II facilitated networking and mutual knowledge transfer by offering high-end workshops on significant topics around the keeping of NHP for research purposes or advanced technology in primate research, not only for its consortium members, but also outreaching to industrial stakeholders and researchers outside Europe. Important collaborations have been initiated that show how important animal welfare issues are also to these groups and joint efforts will contribute to advancement of animal welfare and thus biological and biomedical science beyond the state of the art. Last but not least the workshops provided opportunities for exchange on state-of-the-art technologies, contributing on the best research with NHP in Europe.
EUPRIM-Net II not only addressed scientists but outreached also to non-professionals. This is of particular importance because for the common public it may be difficult to understand why and how NHP are used in experiments and to what extent they may suffer distress during procedures. In the virtual tour through a European Primate Centre that has been produced by EUPRIM-Net II one can learn about research with NHP and how the animals are kept, with the aim to increase acceptance of NHP research among European citizens (http://www.dpz.eu/en/info-center/media-center/virtual-tour.html(se abrirá en una nueva ventana)).
Especially in times of European Citizen’s Initiatives and Petitions against research with animals, it is important to disseminate solid information on NHP research and how animals are kept and handled. Owing to this climate, EUPRIM-Net II has hosted a workshop “Alternative methods for the use of non-human primates in biomedical research” bringing together key scientists working on alternatives to animal research with experts from governmental and non-governmental organisations. During this workshop parallel sessions were organised to stimulate discussion on the challenges of advancing the use of alternative methods for NHP. Subgroups voted on four statements and together composed a list with opportunities and priorities regarding implementation of all 3Rs alternatives, stem cells, NHP in vivo models, and communication. The white paper that resulted from of the Workshop [ALTEX (31)4: 520-529; DOI: http://dx.doi.org/10.14573/altex.1406231](se abrirá en una nueva ventana) can serve as guiding document for future activities.
Education. Article 28 of the Directive on the protection of animals used for scientific purposes (2010/63/EC) requires the Member States to ensure “that staff be adequately educated, trained and competent”. Even before the revision of the European Directive, EUPRIM-Net II had designed a strong programme for continued professional development of staff working with and on NHP. Workshop-like courses on different topics with international experts offered high-end education for scientists and veterinarians, while moreover courses were offered in their local language to animal trainers and care takers as well as technicians, in many instances a rare opportunity for them to receive customised education. Moreover, these courses have represented a very important opportunity for Continued Professional Development (CPD) for all 335 participants from ten different European and four non-European countries and have given NHP staff a chance to exchange with colleagues without the fear of resentments which they may encounter at mixed events.
Developed from scratch was a NHP-specific course meeting FELASA (Federation of laboratory animal science associations) guidelines for the accreditation of laboratory animal science (LAS) courses. This course comprises theoretical and practical contents and a comprehensive E-Learning part was developed especially for this course. The course will probably be accredited before the end of this year (2015) and will then be the first NHP-specific course in Europe certified by FELASA, revolutionising basic education of new staff working with NHP and contributing to harmonisation of NHP-staff education in Europe.
Thirdly, a EUPRIM-Net Seminar Group was formed with participation of ethologists, veterinarians, animal trainers and scientists who created and now offers eight standardised lectures on different topics around NHP keeping and handling to NHP-keeping facilities as in-house trainings (www.euprim-net.eu/network/ABM-seminars.html). These trainings are extremely important because the EUPRIM-Net expert inspects the on-site conditions and can customise the training according to the needs of the facility. Even beyond the EUPRIM-Net II term, Seminar Group members have been asked to give trainings to academic and industrial laboratories.
BioBanking has a high merit for the 3Rs as the sharing of tissues and organs implies the reduction of experimental animals sacrificed for scientific purposes. Over its 54 months duration EUPRIM‐Net II has provided 97 organ‐ and tissue samples of NHP to 21 research projects. All in all, the EUPRIM‐Net Genebanks provided 80 samples that were funded by the European Commission via EUPRIM‐Net II to 17 research projects. Since maximally 20% of the total infrastructure can be subsidised by the EU that means that at least 485 organ‐ and tissue‐samples (actually, close to 600 samples are more likely) and at least 400 gene samples have been distributed by the EUPRIM‐Net institutes involved. These samples have contributed to progress of European research, at the same time sparing animals that would otherwise have been sacrificed especially for the research project in question. Apart from the number of distributed samples it should be emphasised that the EUPRIM-Net BioBank is a unique and valuable resource in Europe for access to characterised, high quality NHP biomaterial used by researchers in biological and biomedical research accelerating progress in the solution of biomedical and other scientific problems that depend on supply of NHP biomaterial.
PRIMOCID. In some cases NHP is the only appropriate animal model that can give answers to specific scientific questions. Experimentation with NHP requires expert knowledge and specialised facilities. Through PRIMOCID (Transnational Access to Non-Human Primate Models of Chronic Immune Disorders) the infrastructure project EUPRIM-Net II has offered expert knowledge, facilities and animals to European researchers. Over the 54 months eight projects have been funded by the European Commission. Since maximally 20% of the total infrastructure can be subsidised that means that at least 40 excellent projects in the fields of Multiple Sclerosis, Tuberculosis, Malaria, Alzheimer Disease, DNA vaccines, arthritis, behaviour and more have been conducted that would not have been possible without the kind of expert service offered by the EUPRIM-Net consortium, or only under conditions not optimal for the research or the animals.
Tools to measure Infection and Disease in NHP. When working with NHP, there is always the possibility of anthropo-zoonotic transfer from humans to NHP and vice versa. Moreover, the 3R concept implies that more data are obtained from minimal numbers of animals, and from small samples that are preferably collected using non- or less-invasive sampling techniques. Particularly, New World monkeys (NWM), like marmosets and tamarins, are progressively used in biomedical research. A serious drawback of using NWM is the limited blood sample volume that can be collected. Flow cytometric methods (e.g. Luminex platform), or the analysis of cellular transcripts using microarrays or quantitative RT-PCR assays were developed during the EUPRIM-Net II term to enable researchers to analyse multiple parameters from a single small sample. Here, specific emphasis was laid on the development and application of the above mentioned technologies to monitor biomarkers in NWM and macaques. The implementation of the new technologies in NHP research has significantly contributed to the 3R principle. Use of archival tissue samples, or non-, or less-invasively obtained samples further contributed to the 3Rs, enhancing 1) the quality of life of the animals used in research, and 2) responding to the increasing demand of data by scientists. Importantly, all techniques established in the course of this research project can easily be adapted and applied to other pathogens or diseases of NHP.
Monkey “home office”. Induced stress is a confounding factor for many research questions. Reduction of stress contributes to the refinement of studies by improving the quality of the collected data. As a consequence the number of animals needed for a study is reduced and animal welfare in experimental conditions is improved. An important finding during the EUPRIM-Net II term was that cage-based testing can have an enrichment effect in socially healthy groups in housing colonies of baboons, which may be considered to be the least stress imposing setting investigated here. In a laboratory environment, using cage-based approaches compared to lab-based approaches including animal handling, marmoset monkeys preliminarily show lower acute stress responses. Yet, lab-based approaches with regular handling in marmosets did not lead to sustained increased stress levels. Results were partly obtained from different NHP species and may not transfer to other species. But if cage-based approaches also reduce stress in other NHP species and settings then the cage-based behavioral testing and automated training procedure developed for rhesus monkeys in neuroscientific context could mark a first step towards a refined experimental approach in this field.
Development of in vitro (alternative) technologies. European citizens call for the use of alternative methods replacing animal experiments and testing. In reality, molecular or cell-culture-based methods need validation that is time-consuming. Nevertheless, the development of in vitro methods is a refinement with high potential to reduce the number of animals needed or to even replace an animal experiment and should be pursued. Sometimes an in vitro method can help to alleviate discomfort for the animal undergoing experimental procedures. For these reasons in vitro technologies should be expedited.
Biomedical research that focuses on the central nervous system (CNS) in general goes accompanied with moderate to severe discomfort for animals involved in such research. Still relatively large numbers of NHP are used in in vivo CNS studies, and there is an urgent need to develop alternative in vitro models for NHP CNS studies. The methods developed during the EUPRIM-Net II term are all initiated from ‘left-over’ material from other experiments. We are currently applying these techniques as a pre in vivo test phase, enabling colleagues with valuable information leading to a reduction of animals necessary before the start of in vivo experiments. Not only does this lead to better science with fewer animals, it also makes more complete use of the animals from which the cultures are initiated.
HIV vaccine research requires NHP to be kept under experimental conditions for extended periods of time. The in vitro methods developed within EUPRIM-Net II have the potential to contribute to refinement in HIV vaccine research by reducing the time the animal has to spend in the experimental setting and by helping to distribute animals evenly into the various experimental groups (as means of standardisation) which may even reduce number of experimental animals without losing statistical significance.
Adjuvants are enhancing the immunisation effect but may also cause discomfort to humans but also to research animals in the form of unwanted side-effects. In EUPRIM-Net II in vitro assays were developed to both identify new adjuvant candidates with good adjuvating potential as well as to characterise the granuloma-inducing potential of new adjuvant candidates.
Scientific evaluation of Animal Behavioural Management (ABM) and Positive Reinforcement Training (PRT). Adaptation and training of laboratory primates for participation in procedures are generally accepted as methods to reduce the stress level for the animals and promote more reliable experimental results. However, there is a limited amount of scientific literature within this area and thus little solid information on the effectiveness of measures such as ABM or PRT. EUPRIM-Net II contributed with research in this area and seven articles and a book chapter have been published or are in preparation.
List of Websites:
Contact:
Prof. Stefan Treue (Project Coordinator)
Dr. Björg Pauling (Project Management)
German Primate Center (DPZ)
Kellnerweg 4
37077 Göttingen, Germany
Tel.: +495513851454
FAX: +495513851228
Email: euprim@dpz.eu
URL: www.euprim-net.eu
List of Partners:
Deutsches Primatenzentrum GmbH (DPZ), Göttingen, Germany
Biomedical Primate Research Centre (BPRC), Rijswijk, Netherlands
Department of Health (DH) with
Public Health England (PHE), Porton Down, United Kingdom &
Medicines and Healthcare products Regulatory Agency (MHRA) with
National Institute for Biological Standard and Control (NIBSC), South Mimms, United Kingdom
Westfälische-Wilhelms-Universität (WWU) with
Centre for Reproduction and Andrology (CeRA), Münster, Germany
Consiglio Nazionale delle Ricerche (CNR) with
Institute of Cellular Biology and Neurobiology (IBCN), Rome, Italy
Université de Strasbourg (UdS) with
Centre de Primatologie (CdP), Niederhausbergen, France
Centre National de la Recherche Scientific (CNRS) with
Station de Primatologie (SdP), Rousset, France
Medical Research Council (MRC) with
Centre for Macaques (CFM), Porton Down, United Kingdom
Karolinska Institutet (KI) with
Astrid-Fagraeus Laboratory, Solna, Sweden
