Laboratory Animals the INTERNATIONAL JOURNAL of LABORATORY ANIMAL SCIENCE, MEDICINE, TECHNOLOGY and WELFARE

Volume 53 Number 5 October 2019 ISSN 0023-6772

Laboratory Animals THE INTERNATIONAL JOURNAL OF LABORATORY ANIMAL SCIENCE, MEDICINE, TECHNOLOGY AND WELFARE

Official Journal of AFSTAL, ECLAM, ESLAV, FELASA, GV-SOLAS, ILAF, LASA, NVP, SECAL, SGV, SPCAL

Published on behalf of Laboratory Animals Ltd. journals.sagepub.com/home/lan by SAGE Publications Ltd.

Volume 53 Number 5 October 2019 Contents

Working Party Report Revised recommendations for health monitoring of non-human primate colonies (2018): FELASA Working Group Report 429 I Balansard, L Cleverley, KL Cutler, MG Spa˚ngberg, K Thibault-Duprey and JAM Langermans

Original Articles Characterization of inflammatory infiltrate of ulcerative dermatitis in C57BL/6NCrl-Tg(HMGA1P6)1Pg mice 447 D De Biase, F Esposito, M De Martino, C Pirozzi, A Luciano, G Palma, GM Raso, V Iovane, S Marzocco, A Fusco and O Paciello

Comparison of pre-emptive butorphanol or metamizole with ketamine þmedetomidine and s-ketamine þ medetomidine anaesthesia in improving intraoperative analgesia in mice 459 C Bauer, U Schillinger, J Brandl, A Meyer-Lindenberg, A Ott and C Baumgartner

Acinetobacter species in laboratory mice: species survey and antimicrobial resistance 470 L Benga, AT Feßler, WPM Benten, E Engelhardt, K Ko¨hrer, S Schwarz and M Sager

Reliability of behavioral tests in the middle cerebral artery occlusion model of rat 478 J Yu, J Moon, J Jang, JI Choi, J Jung, S Hwang and M Kim

Effect of three opioid-based analgesic protocols on the perioperative autonomic-mediated cardiovascular response in sheep 491 L Bellini and GM De Benedictis

A REDCap application that links researchers, animal facility staff and members of the IACUC in animal health monitoring 500 C Carvajal, C Vallejos, D Lemaitre, J Ruiz, C Guzma´n, V Aguilera, D Ban˜o and SD Calligaris

Short Report Implementing strategies to reduce singly housed male mice 508 G Azkona and JM Caballero

News Watch out for three FELASA Working Group recommendations 513 J-P Mocho

Calendar of events/Index to advertisers 524 LAJ worth its weight in GOLD

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www.marshallbio.com Working Party Report Laboratory Animals 2019, Vol. 53(5) 429–446 Revised recommendations for health ! The Author(s) 2019 monitoring of non-human primate colonies Article reuse guidelines: sagepub.com/journals- permissions (2018): FELASA Working Group Report DOI: 10.1177/0023677219844541 journals.sagepub.com/home/lan

Ivan Balansard1, Lorna Cleverley2, Keith L Cutler3, Mats G Spa˚ngberg4, Kevin Thibault-Duprey5 and Jan AM Langermans6,7

Abstract The genetic and biological similarity between non-human primates and humans has ensured the continued use of primates in biomedical research where other species cannot be used. Health-monitoring programmes for non-human primates provide an approach to monitor and control both endemic and incoming agents that may cause zoonotic and anthroponotic disease or interfere with research outcomes. In 1999 FELASA recommendations were published which aimed to provide a harmonized approach to health monitoring programmes for non-human primates. Scientific and technological progress, understanding of non-human primates and evolving microbiology has necessitated a review and replacement of the current recommendations. These new recommendations are aimed at users and breeders of the commonly used non-human primates; Macaca mulatta (Rhesus macaque) and Macaca fascicularis (Cynomolgus macaque). In addition, other species including Callithrix jacchus (Common marmoset) Saimiri sciureus (Squirrel monkey) and others are included. The important and challenging aspects of non-human primate health-monitoring programmes are discussed, including management protocols to maintain and improve health status, health screening strategies and procedures, health reporting and certification. In addition, information is provided on specific micro-organisms and the recommended frequency of testing.

Keywords husbandry, care, laboratory animal welfare, quality assurance/control

Date received: 2 November 2018; accepted: 10 March 2019

Introduction

Since the publication in 1999 of recommendations for 1 1 Centre d’Exploration Fonctionnelle et de Formation, Campus health monitoring of non-human primate colonies sci- Me´decine Sante´, Marseille, France ence has evolved. New methodologies have been devel- 2Marshall Bioresources, Hull, UK oped and priorities for health screening non-human 3Endell Veterinary Group, Salisbury, UK 4 primates (NHPs) have changed with new assessments Astrid Fagræus Laboratory, Karolinska Institute, Sweden 5Animal Models & Bioimaging, Sanofi Pasteur R&D, France outlined in EU directive 2010/63/EU. This has necessi- 6Animal Science Department, Biomedical Primate Research tated a review of the current health-monitoring guide- Centre, The Netherlands lines for NHPs. 7Department of Animals in Science & Society, Faculty of Veterinary The use of NHPs in biomedical research is still Medicine, Utrecht University, The Netherlands necessary for the advancement of science.2 This use, Corresponding author: however, should be limited to investigations where Jan AM Langermans, Biomedical Primate Research Centre, Lange alternative methodologies and where in-vitro models Kleiweg 161, Rijswijk, 2288GJ, The Netherlands. or other animal models are unavailable, for the Email: [email protected] 430 Laboratory Animals 53(5) preservation of the respective NHP species and in Identification of the microbiological unit is key to essential areas of biomedical research with a high prob- health monitoring and management. ability of achieving benefit for human health. Pathogenic agent: an infectious or parasitic agent The source of NHPs in biomedical research is not capable of causing clinical disease and possibly mortal- limited to European breeding colonies, although it is ity in immunocompetent animals. now effectively limited to purpose-bred animals. In Opportunistic agent: an agent which may be present EU member states, only NHPs that are offspring of within the animal’s environment or as part of the animals that have been bred in captivity (F2/F2þ gen- normal commensal flora without causing clinical dis- eration bred) will be permitted to be used after 10 ease but may become pathogenic in certain pathological November, 2022. Monitoring the health and improving conditions. Opportunist organisms may act synergistic- the welfare of these animals at all stages, in both the ally with other infectious agents or in situations when source breeding colonies and in the research centres immune status is compromised. through a harmonized approach to husbandry and Interfering agent: an agent which may or may not health monitoring, will ensure the suitability of NHPs have any pathogenic or opportunistic capacity but in quality-driven research. In addition, since many which has the potential to interfere with scientific aims. pathogens have the ability to infect and cause disease Zoonotic agent: a pathogen which is naturally trans- in NHPs and also infect and cause disease in humans, mitted between vertebrate animals and man. this harmonized approach will also assist in reducing Anthroponotic agent: a pathogen which may be the risk of zoonosis to animal handlers and conversely transmitted from man to animals. anthroponotic infections in animals. In contrast to many other commonly-used labora- Management protocols to maintain or tory animal species NHPs pose considerable challenges improve health status when trying to establish and manage health status. They are complex and mostly large animals which The health status of any group of animals is determined must be co-housed in compatible social groups in an by various factors interacting to exert influence, with enriched environment. Gestation and time to weaning the most significant challenge provided by exposure to is prolonged and the reproductive rate is generally slow infectious microorganisms. Maintaining an established compared with other laboratory species. In addition, health status within a colony depends on eliminating the importance of appropriate behavioural manage- challenges from non-endemic infectious agents entering ment and socialization is being increasingly recognized the colony and reducing the level of challenges from in the maintenance of non-human primate health and endemic infectious agents. The consequence of these welfare, requiring the consideration of human health in challenges is determined by the immune status of indi- addition to animal health. vidual animals and the exposed population as a whole. All NHPs supplied for use in biomedical research There are both direct and indirect influences exerted on should be of known health status, particularly with the immune system associated with environmental con- respect to major zoonotic pathogens such as ditions and management practices. Cercopithecine herpesvirus 1 (Herpes simiae, Herpes B In order to maintain physical and mental health it is virus) and Mycobacterium tuberculosis.3–5 When sup- important to reduce the potential for stress or distress as plied, they must be accompanied by a relevant lifetime these factors are known to have a negative effect on veterinary history and a health declaration detailing all immune function. These effects can be minimized by pro- health screening carried out including when it was car- viding an appropriate and hygienic environment together ried out, the name/details of the laboratory performing with the supply of a wholesome and balanced diet. The the testing and the methodology employed. importance of manipulation, handling and socialization to avoid stress is also an important consideration. Definitions Minimizing the level of challenge from endemic and non-endemic infectious agents should include separ- Microbiological unit: the microbiological unit is con- ation of clinically unwell animals from the colony. sidered as the frame for health monitoring. It is possible Where possible, sick animals should be separated for the microbiological unit to be a single animal but in from the group for treatment to allow recovery and the context of an NHP this is considered unlikely. More improve immune function. likely is the group of animals occupying a room, a set of rooms or a defined area which may be complicated by Endemic infections access to an external environment or by being serviced by the same plant or staff as other groups of animals In order to improve the health status of a colony, which may link the groups in a microbiological sense. sources of endemic infection should be identified and Balansard et al. 431 action taken to reduce or prevent the transmission of example fresh fruit and vegetables can serve as vehicles the infectious agent to other non-infected animals. of transmission. Maintaining the highest possible standards of hygiene and the immediate isolation of infected animals are Health-screening strategies and protocols important, but it is also important to implement appropriate health screening protocols to identify infected Health screening at its most basic level starts with close animals and determine appropriate action. In such situ- observation of every primate by experienced staff at an ations, it is vital that realistic goals are set, and that the appropriate frequency (at least daily). This should also limitations of the available technology, including reli- include periodic clinical examination by a veterinary able animal identification and sensitivity and specificity surgeon familiar with the species in question. Ill of laboratory-testing technology are recognized and health often first presents as behavioural changes understood. before clinical signs of disease become apparent. Behavioural changes including inactivity, hiding away, Non-endemic infections changes in eating or drinking, crouching and scratching excessively can be primary indicators of ill health. For The greatest challenge from non-endemic infectious example, tuberculosis in macacques may present as agents for any group of animals of any species, is under-eating and weight loss without any clinical likely to be as a result of the introduction of additional signs. Unexplained behavioural changes and clinical animals into a colony. If this is necessary, biosecurity signs should be investigated further to establish the precautions to prevent the inadvertent introduction of cause, but it must be considered that NHPs often infectious agents must be carefully considered and mask signs of pain and it may be difficult to identify implemented. This should include a detailed examin- affected NHPs by behavioural changes. ation of the health status of the natal group and Post-mortem examination of animals who die unex- group of origin of the animal(s) in question and a pectedly or are euthanized, either as a planned action or period of quarantine. The duration of quarantine will as an emergency procedure, also provide valuable infor- depend on the particular pathogens of interest. During mation about the health status of individuals and their quarantine, clinical samples should be collected for colony. appropriate laboratory testing and appropriate prophy- The most common understanding of health monitor- lactic or preventative medication given, or vaccination ing is the collection of samples of blood or faeces. carried out as appropriate.6 When designing a health-screening programme the fol- Vaccination can be an important component of dis- lowing factors should be considered: ease-control programmes to minimize the transmission of non-endemic infections in a colony, for example, . What sample types should be collected: blood, some animal facilities have chosen to immunize their faeces, swabs, tissues? macaques with measles vaccine. The limitations asso- . How many animals should be sampled? ciated with vaccination should be considered. . Which animals should be sampled? Vaccination can, for example, reduce the prevalence . How frequently should the animals be sampled? or severity of a disease but may not prevent infection . Which type of analysis is appropriate: PCR, ser- in animals who are unable to sero-convert. In addition, ology, culture, microscopy? vaccination may mask an infection, resulting in the delay of treatment and promoting dissemination to The answers to these questions depend on various fac- unvaccinated subjects. Health-monitoring programmes tors including what is understood about the biology can be hampered because serological analysis to deter- and epidemiology of the organism of interest. mine presence or absence of disease is not easily Consideration should also be given to the aim of achieved in vaccinated primates for the vaccinated screening. Health screening may be used to confirm a agent. However, to some extent this can be overcome negative colony, to determine the presence of an infec- by using direct detection methods such as polymerase tious agent or to determine the prevalence of an ende- chain reaction (PCR). mic agent. Understanding whether the host’s immune A significant risk factor associated with disease response to challenge an infection will eliminate the transmission to NHPs is presented by wildlife, particu- infectious agent without persistent, latent or chronic larly rodents, insects and birds. A vector-control pro- carrier status is also important (Table 1A–1C). gramme should be in place to reduce transmission risk. To establish prior challenge from an infectious agent Animal handlers also present a significant risk as many amongst a group of animals, a single positive antibody organisms associated with human disease are also able ELISA (enzyme-linked immunosorbent assay)-test to infect NHPs. Food may also present a risk, for result can do this, but the agent may no longer be 432 Laboratory Animals 53(5)

Table 1a. Recommended infectious agents to monitor and Table 1b. Recommended infectious agents to monitor and frequencies of monitoring for laboratory macaques. frequencies of monitoring for laboratory baboons.

At At least At At least arrival annually arrival annually

Viruses Viruses Rabies, Lyssavirus (a, b) x Rabies, Lyssavirus (a, b) x B virus (Herpesvirus simiae, x x (b) Retroviruses (SIV, STLV, SRV) (b, c) x Cercopithecine herpesvirus 1) Bacteria Filoviruses (Ebola-Reston) x (c) Mycobacterium x x (d) Measles, Morbillivirus x (d) x (d’) – africanum Retroviruses (SIV, STLV, SRV) (e) x – bovis Bacteria – tuberculosis Mycobacterium x x (f) Salmonella spp. xx – africanum – typhimurium – bovis – enteritidis – tuberculosis Shigella spp. xx Salmonella spp. xx Yersinia xx – typhimurium – pseudotuberculosis – enteritidis – enterocolitica Shigella spp. xx Parasites x Yersinia xx Entamoeba histolytica xx – pseudotuberculosis Giardia spp. xx – enterocolitica All Helminths including x x Parasites – Strongyloides stercoralis Entamoeba histolytica xx – Trichuris Giardia spp. xx – Echinococcus multilocularis (e) All Helminths including x x (a) In endemic countries, vaccination could be considered. – Strongyloides stercoralis (b) Except for animals from virus-free closed colonies. – Trichuris (c) It may be necessary to renew this test as a negative result is not – Echinococcus always predictive for retroviruses. multilocularis (g) (d) In case of closed colonies bred in Europe, and under vet supervision, TB management can be alleviated by checking less often or (a) In endemic countries, vaccination should be considered. only on a representative sample of the population. (b) Except for countries which are officially free from this disease (e) only in outdoor facilities, in endemic areas. and for closed colonies with NHP coming from these countries. Other infectious agents will be added if necessary considering the (c) Only for cynomolgus coming from Philippines. national regulation, the origin of the animals, the type of research (d) Except if vaccinated (d’) can be usefull in outdoor facilities protocol to be performed and the risk assessment. (contact with humans). (e) It may be necessary to repeat this test as a negative result is not always predictive for retroviruses. If testing is used to determine prevalence, it may be (f) In case of closed colonies bred in Europe, and under vet super- sufficient to sample and test a proportion of the animals vision, TB management can be alleviated by checking less often or in the group of interest. only on a representative sample of the population. (g) only in outdoor facilities, in endemic areas. It is important that sampling is representative, test- Other infectious agents will be added if necessary considering the ing appropriate animals (e.g. by age or gender) and the national regulation, the origin of the animals, the type of research number of animals to be tested must be determined protocol to be performed and the risk assessment. using valid statistical methods (Table 2). Understanding the epidemiology, and particularly the rate of transmission between individuals, of the present. If determining that a colony is negative for a infectious agent under consideration is essential. An particular agent, then demonstrating that every animal understanding of epidemiology for each agent will within the group of interest remains seronegative even also influence frequency of sampling and testing. For on multiple occasions may not be sufficient. example, understanding that organisms may be inter- Seronegative latent carriers may exist in which case mittently shed is particularly pertinent when screening additional testing may be required, for example PCR. animals for the presence of enteric pathogens, for Balansard et al. 433

Table 1c. Recommended infectious agents to monitor and (a positive result becomes more likely to be a ‘false’ frequencies of monitoring for laboratory New World positive as the prevalence decreases). When testing to Monkeys (Saimiri sciureus, Callithrix jacchus). assess prevalence, an understanding of predictive value At At least is required to determine the number of animals that arrival annually should be sampled within a population.

Viruses Health-monitoring reports and Rabies, Lyssavirus (a, b) x certification Bacteria Mycobacterium x Standardization of health-monitoring reports and certi- – africanum fication is important to prevent confusion. In order for a – bovis realistic risk assessment, reports and certificates should – tuberculosis provide clarity about whether they relate only to the animals identified on the report, or if they relate to the Salmonella spp. xx source of those animals. Test-negative animals sourced – typhimurium from a colony in which the infectious agent of interest is – enteritidis endemic should be regarded as a higher risk than test- Shigella spp. xxnegative animals from a test-negative source. Yersinia spp. xx The report or certificate should also provide infor- – pseudotuberculosis mation about the laboratory performing the testing – enterocolitica with regard to their accreditation and quality assur- Parasites x ance. Reports should provide information about the Entamoeba histolytica xxmethods employed including published sensitivity and Giardia spp. xxspecificity data where possible. If reporting or certifica- All Helminths including x x tion relates to groups of animals, the numbers of ani- – Strongyloides stercoralis mals sampled and tested relative to the population of the group, the frequency of testing and how these are – Trichuris selected should also be reported, in order to assess the – Echinococcus confidence that can be placed in the conclusions drawn multilocularis (c) from the results. (a) In endemic countries, vaccination could be considered. All facilities that breed or house NHPs for biomed- (b) Except for NHP from countries which are officially free from ical research should implement risk-based programmes this disease. (c) only in outdoor facilities, in endemic areas. for the detection and control of potential pathogens Other infectious agents will be added if necessary considering the and other agents of interest. Where appropriate these national regulation, the origin of the animals, the type of research should be aimed at the eradication of the agent in ques- protocol to be performed and the risk assessment. tion, or prevent its introduction, to ensure the health of animals and staff and also to prevent compromise to example, the collection of rectal swabs for culture on a research programmes. It must be accepted, however, single occasion or daily on three consecutive days. that the aim of eliminating all infectious agents from Consideration should be given to the possible limi- colonies of NHPs may not be a realistic goal nor, in tations of the laboratory-testing technology and meth- fact, desirable. ods employed in health screening (Table 3). Sensitivity and specificity are functions of the testing technology Infectious agents of importance and define the ability of a test to truly identify status. Each of these can be altered, to some extent, usually at This paper considers the species of NHPs most com- the expense of the other, depending on what is required monly used in biomedical research: Macaca mulatta to truly define status, by altering cut-off values. This (Rhesus macaque) and Macaca fascicularis may be done, for example, if there is a desire to eradi- (Cynomolgus macaque). Additional comments relevant cate an infectious agent from a group of animals rather to Callithrix jacchus (Common marmoset), Saimiri than when monitoring prevalence. Predictive value, sciureus (Squirrel monkey), Chlorocebus aethiops rather than being a function of the test, is a function (Vervet monkey) and Papio sp. (Baboons) are included of the prevalence of an agent within a population; a where species specific examples or discussion is con- high prevalence confers a high positive predictive sidered necessary. value (a positive result is likely to be correct) but a The organisms of importance discussed are limited low prevalence confers a low positive predictive value to the major pathogenic, zoonotic and interfering 434

Table 2. Interpretation considerations of standard screening methods used in Animal Health Monitoring.

Serology PCR Culture Microscopy Interpretation

Positive Negative May indicate a previous infection that has now resolved and the organism is no longer being shed. Can be a result of cross-reactivity of antigens used in the serology assay. Confirm assay specificity The sampling protocol may not be sufficient to detect organisms of low prevalence Negative Positive May indicate that the infection is at a early stage prior to seroconversion. PCR specificity is dtermined by the specificity of the primers/probes used. Confirm specificity of the assay. Repeat testing after 2-3 weeks to allow seroconversion time Negative Positive A culture positive result should also give a positive PCR result as both methods rely on a direct detection. A delay in collecting the PCR sample may result in a sample being taken after the infection has resolved giving a negative result. The organism of interest may be intermittently shed and was not present in the PCR sample Misidentification by culture or the specificity of the primer/probe is inappropriate. Repeat Culture and refer for full identification to a refernce laboratory. Positive Negative PCR has greater sensitivity than culture methods and will detect positives at an earlier stage of infection. PCR specificity is determined by the specificity of the primers/probes used. Confirm specificity of the assay. Negative Positive The organism may be intermittently shed and was not present in the PCR sample Misidentification by microscopy or the specificity of the primer/probe is inappropriate. aoaoyAias53(5) Animals Laboratory Repeat microscopy and refer for full identification to a refernce laboratory. Positive Negative PCR has greater sensitivity than microscopy methods and will detect positives at an earlier stage of infection. PCR specificity is determined by the specificity of the primers/probes used. Confirm specificity of the assay. aasr tal. et Balansard Table 3. The limitations of available test methods and recommended actions after confirming a positive result.

Organisms Tests Available Limitations Purpose Action to be Taken

Cercopithecine Herpesvirus 1, Serology Does not detect latent Virus Quarantine Screening Separate positive animal from Herpesvirus simiae Herpes B non-infected animals. Take action to protect staff from this severe zoonosis Mutiplex Flurorometric Does not detect early disease Annual Health Screening Immunoassay ELISA Assays may cross reacts with HSV-1. Confirm specificity Virus isolation/Culture Not widely available. Requires Identify genotype high containment 3 or 4 depending on country. Identify lesions Polymerase Chain Reaction Confirm negative after animal handler bite Confirmation of serology positive result Simian Immunodeficiency Virus Serology: Does not detect latent Virus Quarantine Screening Depending on the retrovirus, SIV evaluate the risk of contamination to the colony and evaluate risk to research. Simian T-cell Lymphotrophic Mutiplex Flurorometric Does not detect early disease Virus, STLV Immunoassay Simian type D Retroviruses, ELISA 30% non-serocoversion in SRV(D) SRV(D) Western blotting Not widely available Confirmation of serology positive result. Polymerase Chain Reaction SRV D RT-PCR may be serotype Confirmation of serology positive specific ie. gag genes only result detect types 1-3. Monitor Viral load Simian Foamy Viruses - SFV Serology Does not detect latent Virus Quarantine Screening Evaluate the risk to research (Spumavirus) Mutiplex Flurorometric Does not detect early disease Pre-screening for research Immunoassay involving maintenance of cell cultures ELISA ‘ and transplant studies. Western blotting Not widely available Polymerase Chain Reaction Confirmation of serology positive result (continued) 435 436 Table 3. Continued.

Organisms Tests Available Limitations Purpose Action to be Taken

Measles (Paramyxovirus- Serology IgG assays do not detect early Quarantine Screening Morbillivirus) disease Mutiplex Fluorometric Post-vaccination to determine Monitor clinical signs. Evaluate Immunoassay seroconversion risks of contamination of the colony and staff. Evaluate impact on research ELISA Polymerase Chain Reaction Oral swabs and blood. Confirmation of infection

Hepatitis Viruses A, B and C Serology IgG assays do not detect early Quarantine Screening Monitor biochemistry, evaluate disease risks of contamination to the colony and staff. Evaluate risks on research ELISA Post vaccination to determine seroconversion Infection confirmation in unvaccinated groups Rabies (Rhabdovirus, Lyssavirus) Serology Quarantine Screening Fluoresent Antibody Virus Not widely available Post vaccination to determine Separate animal from non Neutralisation seroconversion infected animals. Take action to protect staff from this severe zoonosis Rapid Fluorescent Focus Not widely available Inhibition Test ELISA Does not detect neutralising antibody Polymerase Chain Reaction Confirmation of infection Monkeypox (Poxvirus - Serology Does not detect early disease Quarantine Screening Monitor clinical signs, evaluate Orthopoxvirus) risks of contamination of the colony and staff, Evaluate aoaoyAias53(5) Animals Laboratory impact on research. ELISA MFIA Polymerase Chain Reaction Filoviruses Serology Does not detect early disease Quarantine Screening Separate snimal from non- infected animals. Take action to protect staff from this severe zoonosis. Polymerase Chain Reaction (continued) aasr tal. et Balansard Table 3. Continued.

Organisms Tests Available Limitations Purpose Action to be Taken

Mycobacteria Tuberculin Skin Test Prone to false positives and false Quarantine Screening Separate animal from non- negatives infected aniamsls. Take actions to protect staff from this severe zoonosis. Serology Some animals may not Health Screening seroconvert Mutiplex Fluorometric Does not detect early infection. ImmunoAssay Culture Slow growing. Culture may take up to 6 weeks. Requires tracheal washes. Polymerase Chain Reaction Difficult specimen collection requiring tracheal washes Enterobacteriacae: Culture Variable sensitivity depending on Quarantine Screening Monitor clinical signs. Evaluate method risks of contamination to colony and staff. Evaluate impact on research. Treatment if possible. Salmonella species Shedding in faeces may be Health Screening intermittent Shigella species Yersinia species Campylobacter species Culture Shedding in faeces may be Monitor clinical signs. Evaluate intermittent risks of contamination to staff. Leptospira interrogans Serology Does not detect early infection Monitor clinical signs. Evaluate risks of contamination to the colony and staff. Evaluate impact on research and treatment if possible. Microscopic Aggultination Test Requires paired sera Cross reactivity between serovars ELISA ELISA has poor specificity with high false positives. Culture Only available in specialist laboratories Polymerase Chain Reaction More sensitive than MAT but only Confirm serology positive results detects positive during acute phase Klebsiella pneumoniae Culture Poor sensitivity unless associated Monitor clinical signs. Evaluate with clinical infection risks to staff. 437 Polymerase Chain Reaction (continued) 438 Table 3. Continued.

Organisms Tests Available Limitations Purpose Action to be Taken

Burkholderia pseudomallei Culture Poor sensitivity Monitor clinical signs Polymerase Chain Reaction Not widely available Monitor clinical signs. Evaluate risks of contamination to the colony and staff. Evaluate impact on research Toxoplasma gondii Serology ELISA MFIA Sabin -Feldman Dye Test Gold standard - Only available in specialist reference laboratories Polymerase Chain Reaction Histopathology Intestinal Protozoa Faecal flotation/Microscopy Quarantine Screening Monitor clinical signs. Evaluate Health Screening risks of contamination to the colony and staff. Evaluate impact on research and treatment if possible Formyl-ether concentration/ Poor sensitivity reduced by pool- Microscopy ing samples Faecal Antigen test EIA Variable sensitivity and specificity Rapid Immunochromatographic test Polymerase Chain Reaction Histopathology Intestinal Helminths Faecal flotation/Microscopy Quarantine Screening Monitor clinical signs. Evaluate risks of contamination to the colony and staff. Evaluate impact on research and treat-

ment if possible 53(5) Animals Laboratory Formyl-ether concentration/ Health Screening Microscopy Polymerase Chain Reaction Pneumonyssus simicola Histopathology Post-mortem sampling Monitor clinical signs. Evaluate risks of contamination to the colony. Evaluate impact on research and treatment if possible. (continued) Balansard et al. 439

agents relevant to NHPs from captive bred sources. In specific circumstances it may be important to consider additional agents depending on, for example, where the facility includes an outdoor environment, where there are particular requirements for the research being undertaken or in rare situations where wild-caught animals may be used. risks of contamination tocolony. the Evaluate impact on research and treatment if possible risks of contamination tocolony. the Evaluate impact on research and treatment if possible. Monitor clinical signs. Evaluate Monitor clinical signs. Evaluate Action to be Taken Viruses Cercopithecine herpesvirus 1, Herpesvirus simiae, Herpes B Herpes B virus is arguably the most significant zoonotic infection to consider when working with macaques. Human infection is rare but frequently lethal.7–9 Herpes B virus has been mainly associated with Asian macaques.10 However, Herpes B virus has also been reported in a colony of Capuchin monkeys that developed persistent and asymptomatic Herpes B virus infection, while housed in the proximity of Rhesus macaques Health Screening Purpose Quarantine Screening without direct contact. Other, sometimes fatal, herpes virus infections have been documented in various non- human primate species including Cebus apella, Papio sp. and in Callithrix jacchus.11,12 In macaques, infection is usually transmitted via bites, scratches, sexual contact or contact between saliva and other bodily fluids from an infected animal with defects of the mucous membranes or skin. Vertical transmission can also occur. Viremia is intermittent and does not always coincide with the presence of clinical

weeks signs of infection. Clinical signs are predominantly ves- Fungal cultures may take up to 2 Limitations icular lesions along the muco-cutaneous junctions and in the oral cavity. Peak seroconversion usually occurs between two and three years of age during adolescence and during the onset of sexual maturity. Like other herpes viruses, Herpes B virus exhibits latency, particularly within the trigeminal ganglion. This leads to lifelong infection which may remain qui- escent for considerable periods. During these periods, antibody titres to the initial infection may decrease to a level below the detection limits of serological assays. Titres may rise again periodically, particularly at Culture Tests Available Microscopy times of ‘stress’ or where there is a possibility of immunosuppression. This suggests that a single negative antibody test result, particularly when only an individual animal is being tested, cannot be relied upon to conﬁrm the absence of infection. Absence of this organism is more reliably detected in group situations following serial testing. However, it should be noted that some animals have developed serological evidence of

Continued. infection many years after the establishment and maintenance of a closed, entirely seronegative colony. PCR carried out on swabs collected from the buccal cavity Table 3. Organisms Fungal Infection Ectoparasites may provide additional useful information. A positive 440 Laboratory Animals 53(5) result can only be obtained if the animal is viraemic and and African green monkeys have been documented the virus is being shed at the time of sampling. Direct with STLV infections.13 detection methods such as PCR can be useful to deter- STLV is highly T-cell specific. Transmission is pri- mine the risk of Herpes B virus infection associated marily by the transfer of semen or cervical secretions with potential exposure to animal handlers, for example during breeding. STLV can also be transmitted via after bites or other potential exposure. breast milk to infants. Prevalence increases as animals age and become sexually mature. Immunosuppressive retroviruses Seroconversion is slow, often taking as long as six to nine months even in immunocompetent animals. In Retroviruses are RNA viruses which are characterized individuals co-infected with another immunosuppres- by the presence of the enzyme, reverse transcriptase. sive retrovirus this may be extended to several years. Reverse transcriptase converts single stranded RNA Although there are reports of aetiological links between into double stranded cDNA. In retroviruses, cDNA is STLV infection and malignant lymphomas and lym- integrated into the host-cell genome to make new RNA phoproliferative disease in some primate species, most copies or it remains latent for a period which may be infected animals remain apparently healthy and asymp- measured in years. During this period latently-infected tomatic albeit with possible deleterious effects on the animals can frequently be antibody negative, therefore immune system. sensitivity of this type of testing is limited and a nega- STLV-1 shares between 90 and 95% genetic hom- tive serological result does not confirm the absence of ology with Human T-cell Lymphotrophic Virus-1 infection. Direct methods such as PCR to detect the (HTLV-1) (together referred to as PTLVs – Primate viral genome is more useful in this regard. T-cell Lymphotrophic Viruses). In humans, HTLV-1 Although infections with these viruses appear to is considered the aetiological agent of adult T-cell leu- have minor or absent clinical signs in the animals kaemia, lymphoma and the progressive neurological they infect, the immunosuppression associated with disease Spastic Paresis. Although little has been them increases the morbidity and mortality associated reported, this homology indicates that there may be with other pathogens. These organisms can also have potential for STLV-1 to establish infections in man. significant adverse effects on immunological research, vaccine efficacy or research involving therapies against Simian type D Retroviruses (SRV(D)). The SRV(D) are other retroviruses a group of closely-related viruses which have been isolated from many species of Asian NHPs including macaques. Different serotypes predominate in the dif- Simian Immunodeficiency Virus (SIV). SIV is common ferent NHP species. Infection can result in a wide range amongst populations of African monkeys including of clinical manifestations from a subclinical carrier Chlorocebus aethiops and Papio spp. which generally state to rapidly fatal immunosuppressive disease. remain as asymptomatic carriers of the infection. SIV Direct animal-to-animal contact is the most common is not naturally present in wild populations of Asian route of transmission. macaques. However, these animals can be readily In infected animals SRV(D) can often be demon- infected with SIV making SIV a useful model of HIV strated in a variety of tissues and organs. However, for researchers in this field. The risk of transmission of a significant proportion (30%) of infected macaques SIV in macaque colonies other than those involved in may remain seronegative when tested for SRV(D) spe- HIV research is considered low. Ongoing health screen- cific antibodies. This must be considered when designing for this pathogen is considered unnecessary ing health-screening protocols. except when animals are being sourced for addition to the colony. Simian Foamy Viruses (SFV (Spumavirus)). Foamy SIV carries the potential for zoonotic infection. viruses are highly prevalent in virtually all species of However, very few cases of seroconversion in people NHPs, approaching 100% prevalence in many popula- exposed to this virus have been reported and pathogen- tions. They appear to be non-pathogenic but can inter- icity in humans has yet to be demonstrated. fere with studies requiring the growth or maintenance of cell cultures and transplant studies because of their Simian T-cell Lymphotrophic Virus (STLV). STLV, a highly cytolytic effects. c-type member of the oncornavirus subgroup of retroviruses is endemic in Old World NHPs. Three serotypes Measles (Paramyxovirus: Morbillivirus) have been reported with STLV-A being by far the most prevalent, particularly in macaques, but more than 18 Measles is a highly infectious paramyxovirus of the different species of Old World NHPs including baboons genus Morbillivirus. Humans are the natural host for Balansard et al. 441 this virus, but most non-human primate species, par- for regulatory reasons before animals can be imported ticularly macaques, are susceptible to infection. from countries or regions where rabies remains Infection with measles can have serious consequences endemic. including fatality.14 Measles-virus infections in marmosets can rapidly spread through a colony resulting in Monkeypox (Poxvirus: Orthopoxvirus) high morbidity and mortality.15 Measles infections in colonies of NHP are generally Cases of monkeypox, which is closely related to the result of transmission from animal-care staff. smallpox in man, have been reported in animals Transmission occurs via aerosols, although direct con- from the African continent.18 With the exception tact and fomite transmission may also be possible. of animals sourced from areas where infection is ende- Clinical signs in NHP generally appear approximately mic, it is not considered necessary to screen for this a week following infection and are similar to those seen agent. in man. The predominant signs being a maculopapular skin rash, conjunctivitis, blepharitis and malaise. Filoviruses Measles is immunosuppressive causing transient humoral and cell-mediated immune dysfunction. Historically, cases of infection with Marburg virus and The value of health screening colonies of NHP with strains of Ebola have been reported within captive colo- respect to measles is debatable. The clinical signs of nies of NHPs. Screening for this agent is not considered disease are obvious and positive antibody-test results necessary except in circumstances when animals are confirm previous exposure (or vaccination). sourced from locations where infection is endemic Alternatively, the possible benefits of introducing a vac- (e.g. Africa) and should be performed during quaran- cination policy to protect the animals and confirming tine screening. the animal-care staff are vaccinated, should be New World NHPs, particularly Callithrix jacchus, considered. are well known to have a greater susceptibility to many anthroponotic viral infections than many of the Hepatitis (Orthohepadnavirus and Old World NHPs although there is little evidence for Picornavirus) natural transmission of many of these viruses within primate colonies. Outbreaks of Parainfluenza 1 Historically, Hepatitis viruses including Hepatitis (Sendai-like) virus have, for example, been reported in A and Hepatitis B, have been considered an infection Common marmosets with a relatively high morbidity of humans and the great apes. However, it should be but low mortality.19 borne in mind that there is now one report which demonstrates that naturally occurring transmissible chronic Hepatitis B virus infections can exist among some wild Bacteria 16 Macaca fascicularis populations. In some countries, Mycobacteria Hepatitis B vaccination is routinely carried out on captive NHPs and may be a requirement. Opportunistic infection with various ‘atypical’ A high seroprevalence to simian Hepatitis A, which Mycobacterial species including M. avium and M. intra- may carry the possibility of zoonotic infection, has been cellulare, are frequently diagnosed, particularly in reported in non-human primate colonies.17 Infection is immunocompromised non-human primate species. usually via the faecal–oral route, is self-limiting and However, the Mycobacterial species of greatest con- asymptomatic. Control usually centres on maintaining cern, are M. tuberculosis and M. bovis due to their zoo- appropriate environmental hygiene and the use of per- notic and anthroponotic potential.20,21 Although sonal protective equipment by those in contact with the largely controlled in captive-bred animals in Europe, animals or their environment. these agents remain a significant threat in animals imported from other regions of the world where there Rabies (Rhabdovirus, Lyssavirus) are high rates of human infection or where there is potential for the primates to have contact with endem- In closed colonies of captive-bred NHPs, rabies is not ically infected wildlife. generally of concern. However, a route of transmission Mycobacterial infections are characterized by a pro- is plausible where animals have access to outdoor pens longed incubation period with respiratory disease usu- allowing the possibility of interaction with local ally becoming the most common clinical manifestation wildlife. of infection. Systemic dissemination to almost any In some European countries serological testing and/ organ can occur, as well as latency. Treatment is pro- or vaccination against this zoonotic agent is required longed and is not recommended. 442 Laboratory Animals 53(5)

Regular health screening of animals and personnel is transmission of these organisms to NHP colonies. essential. The frequency of health screening should be Biosecurity measures should also be in place to prevent determined according to risk and should be carried out anthroponotic transmission. on at least an annual basis for all captive NHPs. Salmonellae, including species such as S. typhimur- Screening is commonly based on the tuberculin skin ium and S. enteritidis have the potential to cause gen- test (TST). In NHPs, the test is performed by injecting eralized systemic illness as well as gastro-intestinal either Mammalian Old Tuberculin (MOT) or M. bovis disease. In such situations treatment may be warranted. Purified Protein Derivative (PPD) intra-dermally into However, in less severe situations this may prolong the skin of the eyelid close to its margin, or the hair-free faecal shedding of the organism or even induce a carrier skin of the abdomen or both. The injection site is moni- status. Salmonellosis is a reportable disease in many tored at 24, 48 and 72 hours for the development of a European countries. delayed hypersensitivity response with the degree of Shigellosis is, in many respects similar to erythema and oedema being categorized on an increas- Salmonellosis in NHPs with S. flexneri being the most ing scale. MOT is generally considered the test reagent common species isolated. of choice due to its enhanced ability to evoke an Yersinia enterocolitica and Y. pseudotuberculosis immune response providing improved test sensitivity. have both been associated with disease in colonies of However, this can also result in an unacceptably high NHPs (Y. pestis is not generally considered a risk proportion of false positive results. PPD may be used except possibly in areas where this is endemic in the when MOT availability in Europe is limited. local rat population). Y. pseudotuberculosis in particu- Consideration must be given to the possibility of false lar has been associated with severe infections with sys- negative results which may occur both very early during temic as well as gastro-intestinal manifestations the onset of infection or in the advanced stages of infec- (including reproductive failure) and high mortality tion, if excessive testing leads to anergy or where there amongst groups of marmosets. Vaccination in such is concomitant infection with an immunosuppressive situations should be considered. agent. Screening for the various potential Enterobacterial Given the complexity in diagnosing Mycobacterial pathogens generally relies on microbiological culture. infection, all positive TST results should be investigated Detecting these organisms particularly when screening further to confirm health status. Confirmatory tests apparently healthy populations, can lead to false nega- which could be considered include: tive results. This is due to limitations of the currently available methods and intermittent or low-level excre- . Thoracic radiography, but pulmonary lesions are tion of these organisms. Detection of Enterobacteriacae not specific for tuberculosis and may not be appar- can be improved by careful sample selection (faecal ent very early in the course of the disease. samples are generally regarded as preferable to rectal . Serological or g-interferon testing using blood or swabs), sampling strategy (collecting a rectal swab for serum samples.22–24 culture daily on three consecutive days) will vastly . Culture or PCR testing for M. tuberculosis or increase sensitivity compared to collecting a single M. bovis which can be carried out on sputum, rectal swab for culture, especially when there is inter- broncho-alveolar lavage or necropsy samples. mittent excretion of the target organism. The use of specific enrichment techniques such as Selenite F Although Callithrix jacchus is very susceptible to broth and Chromogenic agars will also improve the experimentally induced infections with M. tubercu- sensitivity of culture. PCR can also be useful to confirm losis25 natural Mycobacterial infections in this species the identity of suspect organisms following culture but appear rare with infrequent reports in the literature. may be considered overly sensitive for use directly on faecal samples. Enterobacteriaceae Before embarking on any screening programme for potentially pathogenic Enterobacteria, consideration NHP are frequently asymptomatic carriers of should be given to any action that may need to be Enterobacteriacae. Enterobacteriacae have the poten- taken should one of the target bacterial species be iso- tial to cause disease, particularly in immunocomprom- lated. This is particularly true if the samples are from ised animals, and they also have the potential for animals where clinical signs are absent. Treatment may zoonotic spread. The bacterial species likely to be of be considered if prevalence is high, however, although greatest interest include Salmonella sp., Shigella sp., this may prevent or reduce the severity of disease or Campylobacter sp. and Yersinia sp.26–28 Pest control reduce bacterial shedding, it may also prolong bacterial policies for insects, rodents, birds and other wildlife shedding and induce a carrier state and is unlikely to are important biosecurity measures in preventing the ever eliminate infection. If treatment is considered Balansard et al. 443 Intestinal protozoal parasites necessary, an antibiotic sensitivity test should be performed to choose an effective antibiotic. A careful This includes Entamoeba histolytica (E. coli and harm-benefit analysis should be performed when treat- E. dispar are considered non-pathogenic), Balantidium ing a whole population to avoid the development of coli and Giardia sp. In low numbers these organisms antibiotic resistance. can be considered part of the normal gut flora, causing no adverse effect but in higher numbers, particularly in Campylobacteraceae immunocompromised individuals intermittent to severe diarrhoea or dysentery may be seen.30,31 Intestinal Many Campylobacter species can be isolated from the protozoal parasites of NHPs carry the risk of zoonotic faeces of clinically healthy and diseased NHPs. The sig- infection. nificance of Campylobacters is debatable and may In apparently healthy animals treatment is not usu- depend on the bacterial species/strain and the present- ally required or recommended but in debilitated ani- ing clinical signs. Treatment is not usually recom- mals treatment with metronidazole can be effective. mended unless the clinical signs are severe. Cryptosporidium parvum infections can occasionally occur in facilities where NHPs have access outside Leptospira interrogans enclosures where puddles of standing water may persist. This agent has zoonotic potential. Various Leptospiral species can infect NHPs and cause Health screening for the presence of intestinal proto- disease, with zoonotic potential, and even death. zoal parasites is generally carried out on pooled faecal Screening is recommended when risk is considered samples using flotation, formyl-ether concentration and highest, in facilities where contact with infected wild microscopy examination although PCR testing is avail- rodents and other wildlife cannot be prevented. able. In most cases, antibiotic treatments are available.

Klebsiella pneumoniae Intestinal helminth parasitism K. pneumoniae is associated with severe infections with Parasitism involving various helminth species including high mortality in marmoset colonies.29 Infections more Strongyloides and Trichuris can affect various species of often present at three months of age when maternal NHP. However, it is rare in animals internally housed antibodies decrease and the individual antibody pro- and fed pelleted diets supplemented with washed fruit duction of the infant is not yet effective. and vegetables. When infection is detected, usually by microscopic examination of pooled faecal samples for Burkholderia (formerly Pseudomonas) the detection of parasite eggs, it is usually without clin- pseudomallei ical signs. Various anthelmintic treatment options are available. B. pseudomallei is an environmental saprophyte found Special consideration should be given to in sub-tropical and tropical regions of mainly South- Echinococcus multilocularis.32 Echinococcus is only East Asia and Australia. In these endemic areas it can considered a risk if primates have access to outdoor easily be isolated from samples of soil and water. enclosures, allowing possible contact with infected Infection resulting in the disease ‘melioidosis’ (mainly foxes in areas where this parasite is endemic. cutaneous lesions) occurs via aerosol transmission or However, Echinococcosis can occur after a long ingestion. Treatment is generally not effective. period of time after importation from an endemic Only animals originating from areas in which this area; this might include several changes of facilities disease is endemic are considered at risk and routine until the illness finally develops. Serological and PCR health screening is not required in European colonies. screening is possible but postmortem examination provides the most useful information.

Protozoal and parasitic infections Pneumonyssus simicola Toxoplasma gondii Lung mites are not considered a signiﬁcant problem in Toxoplasma is only considered a risk where there is European captive NHPs although they can be seen in access by primates to outdoor enclosures allowing pos- macaques imported from regions of the world where sible contact with infected cats. Clinical disease is more they are endemic.33 Coughing is a common clinical severe in new world primates than old world primates sign of Pneumonyssus infection. Diagnosis is usually and primary infection may be fatal. Toxoplasma has made at postmortem and often coincidental. zoonotic potential. Treatment with eﬀective endectocides is available. 444 Laboratory Animals 53(5)

Ectoparasites human primates in biomedical research, production and As in other mammals, mites and lice are a theoretical testing of products and devices (update 2017)’, http://ec. risk in NHP. Especially in outdoor facilities, an ecto- europa.eu/environment/chemicals/lab_animals/pdf/ parasiticide programme should be implemented. Scheer_may2017.pdf (2017). 3. Morton WR, Agy MB, Capuano SV, et al. Specific pathogen-free macaques: definition, history, and current Fungal infections production. ILAR J 2008; 49: 137–144. 4. Bailey C and Mansfield K. Emerging and reemerging Dermatophytosis, usually caused by Trichophyton sp., infectious diseases of nonhuman primates in the labora- resulting in areas of roughly circular alopecia with cuta- tory setting. Vet Pathol 2010; 47: 462–481. neous inflammation around the border will normally be 5. Yee JL, Vanderford TH, Didier ES, et al. Specific patho- detected during routine clinical examination. The diag- gen free macaque colonies: a review of principles and nosis can be made by collecting skin scrapings for recent advances for viral testing and colony management. fungal culture or microscopic examination. J Med Primatol 2016; 45: 55–78. Trichophyton sp. are zoonotic and humans may 6. Roberts JA and Andrews K. Nonhuman primate quar- become infected by direct contact. antine: its evolution and practice. ILAR J 2008; 49: 145–156. 7. Eberle R and Jones-Engel L. Understanding primate Conclusion herpes viruses. J Emerg Dis Virol 2017; 3. It is important to consider that all NHP facilities have 8. Huff JL and Barry PA. B-virus (Cercopithecine herpesvirus 1) infection in humans and macaques: potential for their own specific requirements and that these recom- zoonotic disease. Emerg Infect Dis 2003; 9: 246–250. mendations should be adapted to suit local needs. 9. Tischer BK and Osterrieder N. Herpesviruses: a zoonotic These requirements will depend on the geographical threat? Vet Microbiol 2010; 140: 266–270. location, regulatory requirements, prevalent endemic 10. Weigler BJ, Hird DW, Hilliard JK, et al. Epidemiology of organisms and the research undertaken. cercopithecine herpesvirus 1 (B virus) infection and shed- This review is not exhaustive and continuous efforts ding in a large breeding cohort of rhesus macaques. J to increase the knowledge of infectious diseases of Infect Dis 1993; 167: 257–263. NHPs should be undertaken. Regular monitoring of 11. Matz-Rensing K, Jentsch KD, Rensing S, et al. Fatal scientific developments in this field is required for the Herpes simplex infection in a group of common marmo- optimal management of NHP colonies. We encourage sets (Callithrix jacchus). Vet Pathol 2003; 40: 405–411. other authors to update this guidance as necessary. 12. Wolf RF, Rogers KM, Blewett EL, et al. A naturally occurring fatal case of Herpesvirus papio 2 pneumonia in an infant baboon (Papio hamadryas anubis). JAm Declaration of Conflicting Interests Assoc Lab Anim Sci 2006; 45: 64–68. The author(s) declared no potential conflicts of interest with 13. Ishikawa K, Fukasawa M, Tsujimoto H, et al. respect to the research, authorship, and/or publication of this Serological survey and virus isolation of simian T-cell article. leukemia/T-lymphotropic virus type I (STLV-I) in non- human primates in their native countries. Int J Cancer Funding 1987; 40: 223–239. The author(s) disclosed receipt of the following financial sup- 14. Willy ME, Woodward RA, Thornton VB, et al. port for the research, authorship, and/or publication of this Management of a measles outbreak among Old World article: FELASA. nonhuman primates. Lab Anim Sci 1999; 49: 42–48. 15. Ma¨tz-Rensing K and Bleyer M. Viral diseases of common ORCID iD marmosets. In: Marini RP, Wachtman LM, Tardif SD, et al. (eds) The Common Marmoset in Captivity and Jan AM Langermans https://orcid.org/0000-0002-4806- Biomedical Research. Cambridge, MA: Academic Press, 4412 2019, pp.251–264. 16. Dupinay T, Gheit T, Roques P, et al. Discovery of nat- References urally occurring transmissible chronic hepatitis B virus 1. Weber H, Berge E, Finch J, et al. Health monitoring of infection among Macaca fascicularis from Mauritius nonhuman primate colonies. Recommendations of the Island. Hepatology 2013; 58: 1610–1620. Federation of European Laboratory Animal Science 17. Dogadov DI, Korzaya LI, Karlsen AA, et al. Molecular Associations (FELASA) Working Group on genetic identification of isolates of the hepatitis A virus Nonhuman Primate Health accepted by the FELASA (HAV) from monkeys at Adler Primate Center. J Med Board of Management, 21 November 1998. Lab Anim Primatol 2018; 47: 87–92. 1999; 33: S3–S18. 18. Orba Y, Sasaki M, Yamaguchi H, et al. Orthopoxvirus 2. Scheer (Scientific Committee on Health, Environmental infection among wildlife in Zambia. J Gen Virol 2015; 96: and Emerging Risks). Final Opinion on ‘The need for non- 390–394. Balansard et al. 445

19. Sutherland SD, Almeida JD, Gardner PS, et al. Rapid 26. Wolfensohn S. Shigella infection in macaque colonies: diagnosis and management of parainfluenza I virus infec- case report of an eradication and control program. Lab tion in common marmosets (Callithrix jacchus). Lab Anim Sci 1998; 48: 330–333. Anim 1986; 20: 121–126. 27. Bielli M, Lauzi S, Pratelli A, et al. Pseudotuberculosis in 20. Panarella ML and Bimes RS. A naturally occurring out- marmosets, tamarins, and Goeldi’s monkeys break of tuberculosis in a group of imported cynomolgus (Callithrichidae/Callimiconidae) housed at a European monkeys (Macaca fascicularis). J Am Assoc Lab Anim Sci zoo. J Zoo Wildl Med 1999; 30: 532–536. 2010; 49: 221–225. 28. Zhao N, Li M, Amer S, et al. Mortality in captive Rhesus 21. Matz-Rensing K, Hartmann T, Wendel GM, et al. Monkeys (Macaca mulatta) in China due to infection Outbreak of tuberculosis in a colony of Rhesus with Yersinia pseudotuberculosis Serotype O:1a. Monkeys (Macaca mulatta) after possible indirect con- Ecohealth 2016; 13: 597–601. tact with a human TB patient. J Comp Pathol 2015; 29. Pisharath HR, Cooper TK, Brice AK, et al. Septicemia 153: 81–91. and peritonitis in a colony of common marmosets 22. Bushmitz M, Lecu A, Verreck F, et al. Guidelines for the (Callithrix jacchus) secondary to Klebsiella pneumoniae prevention and control of tuberculosis in non-human pri- infection. Contemp Top Lab Anim Sci 2005; 44: 35–37. mates: recommendations of the European Primate 30. Saleh MN, Thomas JE, Heptinstall JR, et al. Veterinary Association Working Group on Immunologic detection of Giardia duodenalis in a spe- Tuberculosis. J Med Primatol 2009; 38: 59–69. cific pathogen-free captive olive baboon (Papio cynoce- 23. Lyashchenko KP, Greenwald R, Esfandiari J, et al. phalus anubis) colony. J Vet Diagn Invest 2017; 29: PrimaTB STAT-PAK assay, a novel, rapid lateral-flow 916–919. test for tuberculosis in nonhuman primates. Clin Vaccine 31. Zanzani SA, Gazzonis AL, Epis S, et al. Study of the Immunol 2007; 14: 1158–1164. gastrointestinal parasitic fauna of captive non-human pri- 24. Vervenne RA, Jones SL, van Soolingen D, et al. TB diag- mates (Macaca fascicularis). Parasitol Res 2016; 115: nosis in non-human primates: comparison of two inter- 307–312. feron-gamma assays and the skin test for identification of 32. Tappe D, Brehm K, Frosch M, et al. Echinococcus multi- Mycobacterium tuberculosis infection. Vet Immunol locularis infection of several Old World monkey species Immunopathol 2004; 100: 61–71. in a breeding enclosure. Am J Trop Med Hyg 2007; 77: 25. Via LE, Weiner DM, Schimel D, et al. Differential viru- 504–506. lence and disease progression following Mycobacterium 33. Johnson AL, Simonek GD and Keesler RI. Fatal pul- tuberculosis complex infection of the common marmoset monary acariasis in an aged indoor rhesus macaque (Callithrix jacchus). Infect Immun 2013; 81: 2909–2919. (Macaca mulatta). J Med Primatol 2017; 46: 90–92.

Re´sume´ La similarite´ geńe´tique et biologique entre les primates non humains et les humains a assure´ le maintien de l’utilisation des primates dans la recherche biome´dicale ou` d’autres espe`ces ne peuvent pas eˆtre utiliseés. Les programmes de surveillance sanitaire des primates non humains fournissent une approche visant a` surveiller et a` controˆler les agents ende´miques et entrants susceptibles de provoquer des maladies zoono- tiques et anthroponotiques ou d’interfe´rer avec les re´sultats de recherche. Les recommandations FELASA ont e´te´ publieés en 1999. Elles visaient a` mettre en place une approche harmoniseé des programmes de surveillance sanitaire des primates non humains. Les progre`s scientifiques et technologiques, la compre´hension des primates non humains et l’e´volution de la microbiologie ont rendu nećessaire d’examiner et de remplacer les recommandations actuelles. Ces nouvelles recommandations s’adressent aux utilisateurs et aux e´leveurs de primates non humains commune´ment utilise´s;a` savoir le macaque rhe´sus (Macaca mulatta) et le macaque cynomolgus (Macaca fascicularis). D’autres espe`ces sont en outre incluses, dont le Callithrix jacchus (ouistiti commun), le Saimiri sciureus (singe ećureuil) et d’autres. Les aspects importants et difficiles des programmes de surveillance sanitaire des primates non humains sont aborde´s, notamment les protocoles de gestion pour maintenir et ame´liorer l’e´tat de sante´, les strate´gies et proce´dures de de´pistage, les rapports de sante´ et la certification. Des informations sur les micro-organismes spećifiques et la fre´quence des analyses recommandeés sont e´galement fournies.

Abstract Die genetische und biologische A¨hnlichkeit zwischen nicht-menschlichen Primaten und dem Menschen hat fu¨r eine kontinuierliche Verwendung von Primaten in der biomedizinischen Forschung gesorgt, wenn andere Arten nicht verwendet werden ko¨nnen. Gesundheitsmonitoring-Programme fu¨r nicht-menschliche Primaten bieten einen Ansatz zur U¨berwachung und Kontrolle sowohl endemischer als auch eingefu¨hrter Erreger, die 446 Laboratory Animals 53(5)

Zoonosen und Anthroponosen verursachen oder Forschungsergebnisse beeintra¨chtigen ko¨nnen. 1999 wurden FELASA-Empfehlungen vero¨ffentlicht, die einen harmonisierten Ansatz fu¨r Gesundheitsmonitoring- Programme fu¨r nicht-menschliche Primaten bezweckten. Der wissenschaftlich-technologische Fortschritt, das Versta¨ndnis nicht-menschlicher Primaten und die sich entwickelnde Mikrobiologie haben eine U¨berpru¨fung und Aktualisierung der derzeitigen Empfehlungen erforderlich gemacht. Diese neuen Empfehlungen richten sich an Anwender und Zu¨chter der ha¨ufig verwendeten nicht-menschlichen Primaten Macaca mulatta (Rhesus Macaque) und Macaca fascicularis (Cynomolgus Macaque). Des weiteren betreffen sie Arten wie Callithrix jacchus (Weißbu¨schelaffe), Saimiri sciureus (Gewo¨hnlicher Totenkopfaffe) und andere. Es werden wichtige und schwierige Aspekte der Gesundheitsu¨berwachungs- Programme fu¨r nicht-menschliche Primaten diskutiert, darunter Managementprotokolle zur Aufrechterhaltung und Verbesserung des Gesundheitszustandes, Strategien und Verfahren zum Gesundheitsscreening, Gesundheitsberichterstattung und Zertifizierung. Daru¨ber enthalten sie Informationen u¨ber bestimmte Mikroorganismen und die empfohlene Ha¨ufigkeit von Tests.

Resumen La similitud gene´tica y biolo´gica entre los primates no humanos y los humanos ha garantizado la continuidad del uso de primates en la investigacioń biome´dica cuando no se pueden utilizar otras especies. Los programas de control de la salud de los primates no humanos ofrecen un me´todo para controlar y supervisar los agentes ende´micos y entrantes que pueden provocar enfermedades zoono´ticas y antropono´ticas ası´ como interferir en los resultados de las investigaciones. En 1999, se publicaron las recomendaciones de FELASA con la intencioń de presentar un me´todo harmonizado para los programas del control de la salud de primates no humanos. El progreso cientı´fico y tecnolo´gico, junto con el entendimiento de los primates no humanos y los avances en la microbiologıá han hecho que sea necesario una revisioń y un reemplazo de las recomendaciones actuales. Estas nuevas recomendaciones estań destinadas a usuarios y criadores de los primates no humanos ma´s utilizados: Macaca mulatta y Macaca fascicularis. Asimismo, se incluyen otras especies como Callithrix jacchus y Saimiri sciureu, entre otras. Tambień se debaten los aspectos importantes y complejos de los programas para el control de la salud de los primates no humanos como los protocolos de gestioń para mantener y mejorar el estado de salud, procedimientos y estrategias de revisiones me´dicas y certificaciońe informes sobre salud. Finalmente, se facilita informacioń sobre microorganismos especı´ficos y la frecuencia recomendada para la realizacioń de pruebas. Original Article Laboratory Animals 2019, Vol. 53(5) 447–458 ! The Author(s) 2018 Characterization of inflammatory Article reuse guidelines: sagepub.com/journals- infiltrate of ulcerative dermatitis in permissions DOI: 10.1177/0023677218815718 C57BL/6NCrl-Tg(HMGA1P6)1Pg mice journals.sagepub.com/home/lan

Davide De Biase1, Francesco Esposito2, Marco De Martino2, Claudio Pirozzi3, Antonio Luciano4, Giuseppe Palma4, Giuseppina Mattace Raso3, Valentina Iovane5, Stefania Marzocco5, Alfredo Fusco2 and Orlando Paciello1

Abstract Ulcerative dermatitis (UD) is an idiopathic, spontaneous and progressive disease typically affecting C57BL/6 aged mice with an unknown aetiopathogenesis. For this study, we evaluated 25 cases of UD in C57BL/6NCrl- Tg(HMGA1P6)1Pg mice. Formalin-fixed, paraffin-embedded skin samples were submitted to morphological investigations. Immunohistochemical analysis was performed to characterize and quantify inflammatory cells using CD3, CD45/B220, CD4, CD8 and IL-17 antibodies. Mast cell-bound IgE was investigated by immunofluorescence, whereas serum and cryopreserved skin samples were collected for molecular analysis. Student’s t-test (two-tailed) was performed to assess significant differences between the two groups. Affected skin showed extensive areas of ulceration and diffuse, severe and mixed inflammatory infiltrates. No relevant changes were observed in control mice. Immunohistochemical analysis showed a predominant CD3 þ CD4 þ leukocyte population with fewer CD45/B220 and IL-17 immunolabelled cells and mast cell- bound IgE. Increases in TNFa, IL-1b and Il-6 mRNA expression were observed in the skin of affected animals compared to controls. Serum TNFa and IL-6 did not vary between affected and control mice. Inflammatory infiltrates and cytokine expression were consistent with both Th2/IgE and Th17 differentiation, a typical pattern of a type I hypersensitivity reaction. Overall, our data suggest an allergic-based aetiopathogenesis of UD in C57BL/6NCrl-Tg(HMGA1P6)1Pg mice.

Keywords mice, ulcerative dermatitis, histology, immunohistochemistry, refinement

Date received: 4 May 2018; accepted: 5 November 2018

Introduction 1Department of Veterinary Medicine and Animal Production, Ulcerative dermatitis (UD) is an idiopathic, spontaneous University of Naples Federico II, Italy and debilitating condition found in laboratory mice, typ- 2CNR - Institute of Experimental Endocrinology and Oncology, c/o ically aﬀecting aged C57BL/6 (B6) mice or genetically Department of Molecular Medicine and Medical Biotechnologies, engineered mice (GEM) on a C57BL/6 background.1,2 University of Naples Federico II, Italy 3Department of Pharmacy, University of Naples Federico II, Italy Clinically, UD is characterized by intense pruritus and 4S.S.D. Sperimentazione Animale, Istituto Nazionale Tumori- scratching leading to ulcer formation predominantly on IRCCS-‘‘Fondazione G. Pascale’’, Naples, Italy 3–5 the head, neck or upper thorax. The intense pruritus 5Department of Pharmacy, University of Salerno, Fisciano, Italy and the rapid progression of ulcerative lesions may have severe and devastating eﬀects on animal welfare, often Corresponding author: Orlando Paciello, Department of Veterinary Medicine and Animal resulting in secondary bacterial infections and formation Production, University of Naples Federico II, Via Federico Delpino of scars and skin contractures that may impair species- 1, 80137, Naples, Italy. 1,2,4,6 typical behaviours and mobility. Email: [email protected] 448 Laboratory Animals 53(5)

UD is speculated to be a multifactorial disease,1,7,8 also originally obtained from Charles River. Pups were and several predisposing factors, such as environ- biopsied at weaning and analysed for transgene integra- ment,1,2,4,6,9 diet,1,2,10–12 season,1,2,4,6 age at wean- tion by PCR. ing,1,2,13 alopecia,1,2,4,6 sex,1,2,4,9 immune complex All animals were housed in a standardized non- vasculitis,3 and deficiencies in vitamin A metabolism,6 barrier facility at the Istituto Nazionale Tumori have been implicated in the pathogenesis of this condi- ‘Pascale’ (Naples, Italy). Animal care and use as well tion. The detrimental effects on animal welfare and the as all the experimental procedures were in accordance possible negative consequences on research usually with the Guide for the Care and Use of Laboratory require humane euthanasia of the affected mice. The Animals.16 underlying mechanisms contributing to the initiation, All animal procedures were reviewed and approved development and progression of UD are still poorly by the Institutional Ethical Committee of the Istituto understood. Accordingly, many attempted treatments Nazionale dei Tumori, Fondazione Pascale, Naples, have been unsuccessful.1,2,4 Recently, Adams et al. sug- Italy, in accordance with Italian regulations for experi- gested that the use of toenail trims may be a highly mentations on animals. Mice (1–3) of the same sex were effective treatment option for UD lesions,14 resulting housed in wire-topped plastic cages with static filter in complete lesion resolution with no recurrence in tops (Tecniplast, Buguggiate, Italy) and a red plastic approximately 90% of the mice treated. igloo-style shelter (Tecniplast, Buguggiate, Italy) on To our knowledge, an exhaustive characterization of aspen wood bedding (Scobistre; Mucedola srl, the inflammatory infiltrate in spontaneous UD is still Settimo Milanese, Italy). The mice were housed on a lacking. Our findings revealed several similarities to the 12:12-h light:dark cycle and cages were changed twice features of a cutaneous type I hypersensitivity reaction, a week. Plastic cages and bottles were washed in hot suggesting a possible allergic aetiology for UD in mice. (62–82C) water with detergent–disinfectant solution The purpose of this work is to analyse, thorough and were periodically changed to avoid collateral effects histological, immunohistochemical and molecular on the animals. Mice received a pelleted, autoclavable examinations, UD lesions observed in C57BL/6NCrl- rodent diet (long term maintenance diet, cat. n 4RF18; Tg(HMGA1P6)1Pg mice. Our ultimate goal was to add Mucedola srl, Settimo Milanese, Italy) and acidified some information and missing puzzle pieces regarding water (HCl at pH 3.5) ad libitum. Room temperature the true nature of the inflammatory response associated was maintained between 20.0 and 24.0C, and relative with this condition and thereby provide new, useful humidity was maintained between 55% and 65%. insights into UD pathogenesis. Temperature and humidity were constantly recorded (Poseidon2 3266, HWgroup, Casale Monferrato, Italy). Animals, materials and methods The health status of the animals in the facility was monitored annually based on FELASA Recommendations The affected mice were chosen from an experimental (FELASA Health Monitoring Profile 375M, colony of an inbred strain of C57BL/6NCrl- ENVIGORMS Srl, S. Pietro al Natisone, Udine, Tg(HMGA1P6)1Pg mice that were produced by Italy). Two 5-week-old Hsd:ICR CD-1 female sentinel Esposito and colleagues as a part of a study on the mice were exposed to dirty bedding weekly for 10 weeks role of HMGA1 pseudogenes and cancer.15 before being submitted to the commercial vendor (ENVIGORMS Srl, S. Pietro al Natisone, Udine, Generation and housing of C57BL/ Italy) for serology, parasitology and necropsy (see 6NCrl-Tg(HMGA1P6)1Pg mice Supplemental Table 1 online). The maximal number of cages per sentinel was 24. Briefly, the HMGA1P6 expression plasmid was excised with SalI and HindIII restriction endonucleases by Ante- and post-mortem evaluation cleaving 10 mg of the plasmid. The fragment was purified from SeaKem GTG agarose (avoiding exposure to Mice were routinely examined by the facility veterinar- UV light) using the Qbiogene Geneclean Spinkit, then ian (DDB) at least once a week for general condition, dialysed 24 h against 2 l microinjection buffer (10 mM and any mice presenting with skin lesions underwent a Tris-HCl pH 7.2, 0.1 mM EDTA), and diluted to a con- thorough examination. Data collected from clinical centration of 4 ng/ml. The DNA was injected in three evaluation included sex, age, and a detailed description sessions into C57BL/6N-derived zygotes. For this pur- of skin lesions. In addition, a visual examination of pose, C57BL/6N female mice (bred at PolyGene from pruritus was performed, as indicated by Hampton parents obtained from Charles River) were super- et al.2 UD was diagnosed, when appropriate, on the ovulated at 28–34 days of age and mated in the basis of lesion location (thorax, head and neck) and PolyGene mouse facility to C57BL/6N breeder males, the presence of typical crusts or areas of ulceration. De Biase et al. 449

UD lesions were subsequently scored using a system mice (n ¼ 5, 3 females and 2 males) were used as nega- recently developed by Hampton et al. that considered tive controls. The age of B6 healthy mice was 6 months; scratching number, type of lesion, length of lesion the mean age of C57BL/6NCrl-Tg(HMGA1P6)1Pg and regions affected.2 Clinical scoring was performed healthy controls was 16 months (13–20 1.49 SD). once at the time of diagnosis. When affected mice met The severity of dermal lesions was histologically endpoint criteria (more than 10% of the total body assessed as follows (see Supplemental Table 2):2 (1) surface area affected, debilitating scars and/or lesions mild, focal or few foci of inflammation confined to localized to the face and neck) they were deeply anaes- the superficial dermis; (2) moderate, multiple foci of thetized with 5% isoflurane in 100% oxygen (Fluovac inflammation extending to the deep dermis; (3) severe, Anesthetizing System cod. 726425, Harvard Apparatus, regionally extensive foci of inflammation occasionally Cambridge, UK). Subsequently, intra-cardiac puncture extending to the subcutis; (4) marked, with inflamma- for terminal blood collection was performed using tory foci extending to a larger aspect of the subcutis a 23–25-gauge needle. Following blood collection, compared to the severe lesions. euthanasia was ensured by cervical dislocation accord- Regarding the number of mast cells (toluidine blue), ing to the American Veterinary Medical Association this was calculated as the mean value of five randomly (AVMA) guidelines.17 The same procedure for blood selected high-power fields (magnification 40x) in each collection and euthanasia was followed for 10 healthy group (UD vs healthy controls), as previously control animals. described.18 Over an 18-month period, 25 out of 60 C57BL/ For immunohistochemistry (IHC), 4-lm-thick sec- 6NCrl-Tg(HMGA1P6)1Pg mice (41.6%) with lesions tions were processed with the MACH1 Universal compatible with UD were euthanized, and a full post- HRP Polymer Detection Kit (Biocare Medical LLC, mortem examination including histopathology and Concord, CA) as previously described.19 Briefly, skin molecular analysis of the dermal lesion was performed. tissues were mounted on positively charged glass To exclude the possible presence of ectoparasites, the slides (Bio-Optica, Milan). Antigen retrieval pre- fur of the affected animals was sampled from the scapu- treatments were performed using a heat-induced lar area, the ventral cervical region, the axillary area, epitope retrieval (HIER) citrate buffer pH 6.0 (Bio- the inguinal area, and the dorsal rump. The fur was Optica, Milan, Italy) for 20 min at 98C; peroxide subsequently placed on an adhesive tape and then block was applied for 15 min at room temperature, examined under a light microscope using the 10 and and then the sections were incubated for 30 min with 40 objectives. background sniper (Biocare Medical LLC). The primary antibodies were diluted in phosphate-buffered Histopathology and Immunohistochemical saline (PBS) and incubated overnight at 4 C. analysis Horseradish peroxidase (HRP) polymer was added for 30 min at room temperature. After every step, the Standardized skin biopsies from affected animals com- sections were washed in 0.01 M PBS (pH 7.2–7.4). The prised the area of ulceration and the unaffected adja- reaction was revealed by using 3,30-diaminobenzidine cent epidermis. Samples were preserved in 10% neutral (DAB) chromogen diluted in DAB substrate buffer. buffered formalin (code no. 05-01007Q, Bio-Optica, Finally, sections were counterstained in Carazzi’s haema- Milan, Italy), dehydrated and embedded in paraffin toxylin (code n. 05-06012/L, Bio-Optica, Milan, Italy). (code no. 06-7920, Bio-Optica, Milan, Italy). Tissue Primary antibodies included rabbit monoclonal to CD3 sections were stained with haematoxylin and eosin (ab16669,Abcam, Cambridge, UK) diluted 1:200, rabbit (HE) for morphology, toluidine blue (TB) (code no. polyclonal to CD45/B220 as a marker for B cells T3260, Merck KGaA, Darmstadt, Germany) for mast (ab10558 Abcam, Cambridge, UK) diluted 1:1000,20 cell count and histochemical stains such as periodic rabbit polyclonal to CD4 (NBP1-19371, Novus acid-Schiff (PAS) (code no. 04-130802, Bio-Optica, Biological, Littleton, CO, USA) diluted 1:200, rabbit Milan, Italy), methenamine silver (Grocott) (code no. polyclonal to CD8 (ab203035, Abcam, Cambridge, UK) 04-043823, Bio-Optica, Milan, Italy) and Gram diluted 1:100, and goat polyclonal to IL-17 (Santa Cruz (code no. 04-1008002, Bio-Optica, Milan, Italy), the Biotechnology, Santa Cruz, CA) diluted 1:200. For IL-17 latter two for histological identification of fungi antibody, the slides were developed using the Vectastain and bacteria, respectively. Special histochemical stains Elite ABC goat IgG kit (Vector Laboratories, were performed according to manufacturer’s instruc- Burlingame, CA) following a previously described proto- tion. A total of 10 unaffected skin biopsies from col.18 To test the specificity of staining, two negative C57BL/6JOlaHsd mice (n ¼ 5, all females from controls were simultaneously performed: incubating one ENVIGORMS Srl, S. Pietro al Natisone, Udine, section with PBS, omitting the primary antibody, and the Italy) and from C57BL/6NCrl-Tg(HMGA1P6)1Pg other one with an irrelevant and unspecific IgG. 450 Laboratory Animals 53(5)

To identify the predominant lymphocyte subtype, inflammation, sera were diluted 1:10 with the provided the immunohistochemical scoring was independently diluent 1 (e-Bioscience, San Diego, CA), and the performed by two pathologists (DDB and OP) with a enzyme-linked immunosorbent assay (ELISA, e- concordance rate of 95%. The number of immune- Biosciences, San Diego, CA) was performed according labelled cells was calculated for each section and each to the manufacturer’s instructions. The results are antibody in both groups (UD vs healthy controls) and expressed as pg/ml as previously reported.24 is expressed as the mean value of 10 high power fields. For immunofluorescence staining, the pre-treatment Statistical Analysis steps were the same as those used for immunoperoxi- dase labelling following a previously described proced- Data are presented as the mean standard deviation ure.21,22 The primary monoclonal rat anti-mouse IgE (SD). Statistical analyses between the two groups antibody (RMGE00, Invitrogen, Rockford, USA) was were performed using Student’s t-test (two-tailed) diluted 1:100 in PBS and incubated overnight at 4C. using GraphPad Prism (GraphPad Software, San Slides were washed three times in PBS and incubated Diego, CA). Statistical significance was set at p < 0.05. with a fluorescein isothiocyanate (FITC)-conjugated pure goat anti-rat secondary antibody (Alexa FluorÕ 488 conjugated, A-11029 Thermo Fisher, Rockford, Results IL, USA) diluted 1:100 for 2 h at room temperature. Ante- and post-mortem evaluation Slides were rinsed three times with PBS and cover- slipped using Vectashield mounting medium containing UD was clinically diagnosed in 25 out of 60 C57BL/ 40,6-diamidino-2-phenylindole (DAPI) (H-1200 Vector 6NCrl-Tg(HMGA1P6)1Pg mice (41.6%). The mean Laboratories, Burlingame, CA). age of onset was 16 months (13–20 1,49 SD), with For scanning and photo capture, a laser scanning no significant predilection for sex (11 males and 14 microscope (DM6B Leica, Wetzlar, Germany) was females). We observed a season-associated increase in used. The different frames were scanned separately, UD cases, with a predominance of disease (19/25) with appropriate installation of the optical path for during the late spring and summer (see Supplemental excitation and emission of each scan according to the Figure 1). Affected animals showed intense pruritus manufacturer’s instructions. leading to almost uncontrollable scratching and the formation of the characteristic areas of ulceration asso- RNA extraction and real-time ciated with marked dermal fibrosis. Macroscopically, semi-quantitative PCR lesions varied from coalescing crusts to irregularly shaped areas of ulceration extending mostly to the Total RNA was extracted from skin tissues using dorsal cervical region, dorsal and ventral thorax TRIzol Reagent (Bio-Rad Laboratories) according to (Figure 1). Severe lesions gravely affected the animal wel- the manufacturer’s instructions, and cDNA was synthe- fare, resulting in debilitation and, in some cases, altered sized as previously described.23 Each sample contained posture and mobility. The clinical scoring system for the 1–100 ngcDNA in 2 QuantiTect SYBR-Green PCR ulcerative lesions was applied and resulted as follows: 8% Master Mix and primers, tumour necrosis factor a mild (2/25), 64% moderate (16/25) and 28% severe (TNFa), interleukin 6 (IL6), interleukin 1b (IL1b) (7/25). Final health monitoring of the C57BL/6NCrl- and transforming growth factor beta (TGF-b) Tg(HMGA1P6)1Pg mouse colony reported negative (Qiagen, Hilden, Germany) in a final volume of 50 ll. results for ectoparasites as well as for viral, bacterial The relative amount of each studied mRNA was nor- and fungal agents. malized to GAPDH, and data were analysed, according Splenic lymphoma was diagnosed at post-mortem to the 2DDCT method.23 examination and confirmed by means of histology in 32/60 C57BL/6NCrl-Tg(HMGA1P6)1Pg mice, but TNF- and IL-6 determination in serum only in 3/25 of the UD affected animals (12%). No evidence of systemic disease or relevant pathological alter- Blood samples obtained from mice by terminal intra- ations of tissues or organs were observed in the other cardiac puncture were collected in serum separator affected animals (20/25). Frequently, in the most severe tubes. The serum was separated by centrifugation at cases, we observed moderate splenomegaly and reactive 10,000g for 5 min at 20C (Heraeus Biofuge Pico, lymphadenomegaly of the axillary and cervical lymph DJB Labcare Ltd, England). Serum samples were nodes consistent with Kastenmayer’s observations.4 then aliquoted into Eppendorf tubes and stored at Post-mortem ectoparasite examination of the 80C until further analysis. To evaluate TNF-a and affected animals did not show the presence of infest- IL-6 in mouse serum as markers of systemic ations that could have been responsible for UD lesions. De Biase et al. 451

Figure 1. Ulcerative dermatitis, skin, mouse. Typical presentation of murine ulcerative dermatitis showing focal areas of ulceration with fibrosis on the neck (a), upper and dorsal thorax (b), cervical and posterior areas (c).

Histopathology and IHC 12% marked (3/25). The inflammatory population varied between the different histopathologic grades as Histological examination of formalin-fixed and follows: in mild cases the prevalent population was paraffin-embedded 4-lm sections of clinically healthy composed of 40% mast cells, 10% eosinophils, 5% neu- mice showed no evident histopathological changes trophils, 10% macrophages, 20% lymphocytes and and an absence of dermal inflammatory cells and tolui- 15% plasma cells; in moderate cases the prevalent dine blue-positive mast cells (Figure 2(a) and (b)). inflammatory population was composed of 25% mast Conversely, affected skin revealed extensive areas of cells, 5% eosinophils, 15% neutrophils, 20% macro- ulceration and marked hyperplasia with parakeratotic phages, 20% lymphocytes and 20% plasma cells; in to orthokeratotic hyperkeratosis of the adjacent epider- severe cases the prevalent inflammatory population mis. A diffuse and severe inflammation was observed in was composed of 15% mast cells, 5% eosinophils, the dermis, often dissecting through underlying sub- 20% neutrophils, 10% macrophages, 30% lymphocytes cutaneous structures. The inflammatory population and 20% plasma cells; in marked cases the prevalent consisted mostly of neutrophils, toluidine blue-positive inflammatory population was composed of 10% mast mast cells and lymphocytes with macrophages and cells, 5% eosinophils, 25% neutrophils, 5% macro- eosinophils to a lesser extent (Figure 2(c) and (d)). phages, 35% lymphocytes and 20% plasma cells. A variable degree of fibrosis was also noticed, admixed Immunohistochemical analysis was performed to with the areas of inflammation. In contrast to other further characterize the inflammatory lymphocyte authors,3,6 follicular dysplasia, other follicular abnorm- infiltrates, showing a predominant CD3 þ CD4 þ alities, and primary vascular lesions were not observed leukocyte population with fewer of CD45/B220- and in the affected skin samples. The increase in mast cell Il-17-immunolabelled cells. We rarely observed scat- accumulation was statistically significant in the affected tered, CD8-labelled lymphocytes (Figure 4(a) to (e)). (14.5 2.7 SD) skin compared to healthy controls Interestingly, immunofluorescence revealed the pres- (2.04 0.29 SD) (p < 0.05) (Figure 2(e)). ence of numerous IgE-bound mast cells among the Gram, periodic acid-Schiff (PAS) and methenamine inflammatory infiltrates in the dermis and, in marked silver (Grocott) stains did not reveal the presence of lesions, around blood vessels of the underlying subcuta- bacterial or fungal agents associated with dermal neous muscle and adipose tissue (Figure 4(f)). inflammation, although crusts were occasionally associated with superficial, Gram-positive, coccoid bacteria Molecular findings (Figure 3). The severity of the lesions in affected animals were histologically graded as follows: 16% mild Semi-quantitative RT-PCR detected significantly (4/25), 40% moderate (10/25), 32% severe (8/25) and increased IL-6, TNF-a and IL1b in skin samples from 452 Laboratory Animals 53(5)

Figure 2. Histological examination of skin, mouse. (a) Healthy controls did not show any pathological alterations and (b) showed rare toluidine blue-positive mast cells. (c) Ulcerative dermatitic skin showed an extensive area of ulceration with an adjacent hyperplastic epidermis and a dermal severe and mixed inflammatory infiltrate. (d) In affected skin, there was a high number of toluidine blue-positive mast cells. (e) Mast cell count revealed a significantly increased number in ulcerative dermatitis cases compared to healthy controls (p < 0.05). Bars ¼ 100 lm.

Figure 3. Histochemical special stains on ulcerative dermatitic skin samples, mouse. Gram (a), periodic acid–Schiff (PAS) (b) and methenamine silver (Grocott) (c) did not show the presence of bacterial or fungal agents associated with dermal inflammation. Bars ¼ 100 lm.

Discussion affected animals compared to controls (p < 0.05) while TGFb mRNA did not differ between affected and The aim of this study was to investigate, for the first control animals (Figure 5). There was no significant time, a potential pathogenic mechanism for UD through difference in serum IL-6 or TNF-a in affected mice the characterization of inflammatory infiltrates and cyto- compared to healthy subjects (Figure 6). kine expression of C57BL/6NCrl-Tg(HMGA1P6)1Pg De Biase et al. 453

Figure 4. Immunohistochemical analysis of the inflammatory infiltrate, skin, mouse. Inflammatory infiltrate showed a prevalence of CD3 þ (a) and CD4 þ (b) cells. Very few scattered CD8-positive lymphocytes were observed (arrows) (c). CD45/B220 and IL-17 (d, e) immune-labelled inflammatory cells. (f) Immunofluorescence showing intense immunoreactivity of mast cells for IgE antibody (arrows). Bars ¼ 100 lm. mice that spontaneously developed itchy and ulcerative seasonal pattern and in particular to changes in humid- skin lesions. ity,1,2,4 we suggest that the seasonal peak of environ- Our clinical observations, histopathological and mental allergens such as pollen or fungal spores may be molecular findings indicate (1) intense pruritus, (2) sig- responsible, in our case, for the increased number of nificant prevalence of the disease in late spring and affected animals, in agreement with Kastenmayer’s summer, (3) absence of concurrent infection or parasite observations.4 infestation and (4) inflammatory infiltrate and an Th2 cells play a key role in hypersensitivity reaction increased cytokine expression consistent with Th2/IgE by producing several cytokines (namely, IL-4, IL-5 and and Th17 differentiation. These results, in our opinion, IL-13) that are responsible for the class switching of B share similar features observed in cutaneous type I lymphocytes to IgE production.25–27 IgE is essential for hypersensitivity reactions,25 suggesting a possible aller- the activation of mast cells and the release of mediators gic aetiology for UD in mice. (histamine, TNFa) that are the principal culprits for the Immunohistochemical evaluation of dermal inflam- clinical and pathological manifestation (e.g. prur- matory infiltrates in UD lesions interestingly showed an itus)25,28,29 as well as for the influx of Th2 lymphocytes increase in the number of CD4 þ T cells and and eosinophils into the skin.24 During the acute phase Th17 þ cells and a significant increase in mast cells of inflammation, eosinophils produce cytokines such as displaying an intense immunoreactivity for the IgE IL-6 and IL-1b, supporting a Th17 differentiation.29 antibody. Moreover, cytokine analysis showed that A pivotal but yet still unclear role of Th17 cells in affected skin had increased levels of IL-1b, IL6 and allergy has been implicated in several allergic skin dis- TNFa. The type I hypersensitivity reaction is initiated eases, such as atopic dermatitis or psoriasis.30,31 Most by an environmental antigen (called an allergen) that likely, Th17 cells are involved in a late phase of allergic elicits a Th2 cell-mediated response in susceptible sub- response, promoting tissue fibrosis, chronicity of the jects.25 Since UD incidence has been associated with the inflammatory process, and the evolution of chronic 454 Laboratory Animals 53(5)

Figure 5. Cytokine mRNA expression in skin samples. Graphs showing the expression of TNFa, IL-1b, IL-6 and TGFb in UD mice compared to healthy controls; p-values were determined by independent t-test, and the cytokine levels are presented as the means ( SD). All results were considered statistically significant at p < 0.05.

Figure 6. Cytokine levels in serum. Graphs showing the serum levels of IL-6 and TNFa in UD mice compared to healthy controls; p-values were determined by independent t-test, and the cytokine levels are presented as the means ( SD). All results were considered statistically significant at p < 0.05. inflammatory cutaneous lesions.32 TGF-b expression and mast cell proliferation, and they can also induce did not show any noteworthy differences between UD apoptosis in eosinophils.33 It is very likely that, in UD, lesions and healthy controls. This finding is not surpris- TGF-b signalling is impaired, so it fails to inhibit the ing since TGF-b and its signalling pathways have inflammatory process. TNFa and IL-6 are considered inhibitory effects on B and T cells, IgE production, mediators of inflammatory acute response and serum De Biase et al. 455 analysis of their levels is extremely important for the growth rate and migration of different cell lines, con- diagnosis of several systemic conditions, such as inflam- tributing to tumour development.14,36,37 mation and sepsis.34 Here, we found no differences in The assessment of genetic alterations in UD-affected TNFa or IL-6 level in affected animals compared to mice compared to controls was not the principal pur- controls. These findings may indicate that, in the pose of this study. De Martino et al. analysed the whole affected animals, there is no sign of systemic inflamma- transcriptome of WT and HMGA1P6-transgenic mouse tion and/or that the lesions observed are consistent with embryonic fibroblasts (MEFs) by RNA-seq to identify a chronic phase of the allergic response. the genes regulated by HMGA1P6 expression.38 A small percentage of UD-affected mice had splenic The genome-wide RNA expression profile revealed lymphoma (12%). This finding is not surprising and is that about one hundred fifty transcripts (32 upregulated consistent with Andrews’s observations.3 The small and 116 downregulated) were regulated by HMGA1P6 number of mice with splenic lymphoma does not expression with a significant fold-change variation allow a significant correlation between the presence of (FDR adjusted p-value of 0.05). Among these genes, this neoplasm and ulcerative lesions. suppression of tumourigenicity 2 (ST2) was upregu- This work has also some limitations to be addressed. lated in transgenic mice (data not shown). The ST2 First, intense pruritus and scratching are a distinctive gene and his ligand, interleukin 33 (IL33) have critical and constant feature of the clinical presentation of UD, roles in inflammatory processes, particularly regarding so we have to take into account the presence of an mast cells, type 2 CD4 þ T-helper cells and the produc- inflammatory infiltrate secondary to trauma, such as tion of Th2-associated cytokines, promoting the patho- neutrophils, that may slightly influence the interpret- genesis of Th2-related disease such as asthma, atopic ation of the results, especially in relation to triggering dermatitis and anaphylaxis.39,40 These data need to be events in the development of UD. further and appropriately investigated to confirm a gen- Second, we did not evaluate the IL-4, IL-5 and IL-13 etic component in the plethora of mechanisms respon- levels in skin samples, even though they are responsible sible for UD pathogenesis. for the class switching of B lymphocytes to IgE produc- We cannot assert without any uncertainty that tion. To overcome these issues, we thoroughly charac- the manifestation of UD in this specific genetically terized the immune infiltrate by IHC, demonstrating engineered mouse line is representative of the disease the presence of numerous IgE-bound mast cells process in other mutant strains or wild-type B6 mice. among the inflammatory infiltrates in the dermis and, It is possible that C57BL/6NCrl-Tg(HMGA1P6)1Pg in marked lesions, around blood vessels in the under- mice have a predisposition towards an allergic derma- lying subcutaneous muscle and adipose tissue. titis that is a separate entity from UD with a similar In our opinion, the presence of IgE-bound mast cells presentation (pruritus and significant secondary trauma associated with a Th1/Th2 and Th17 immune response from scratching). However, skin samples from different and an increase in IL-1b, TNFa and IL-6, which inter- transgenic mice with a B6 background housed in other vene in the acute phase of the allergic response, are facilities and showing clinical signs of UD were consistent with an allergic eliciting factor for UD reported to our laboratory. Interestingly, the histo- development. pathological findings observed in this study matched Susceptibility to allergic inflammation has a strong the ones described in scientific literature. For consist- genetic component.25 In a 2-year retrospective study, ency, we decided to exclude the ‘external’ UD cases Kastenmayer et al. reported an overall prevalence of from our study for the different housing conditions UD of 4.1% (55/1352) in mice with a C57BL/6 genetic and experimental sampling. background, whereas no UD lesions were observed Although the causative trigger of UD in mice is still in non-C57BL/6 mice.4 Consistent with that, we not completely understood, our data encourage further showed a high prevalence (41.6%) of spontaneous investigations into the hypothesis that this disease has UD lesions in transgenic mice on a C57BL/6 genetic an allergic aetiopathogenesis, also suggesting the bases background overexpressing the HMGA1P6 pseudo- for a possible treatment. The inflammatory Th2 infil- gene. Therefore, it is conceivable that genetic defects trate and its related cytokines can explain, to a certain are responsible for the development of this disease. extent, the partially positive clinical response associated Pseudogenes are DNA sequences that have high hom- with the use of the vitamin E-fortified diet.5 As a matter ology to the corresponding functional gene but have of fact, vitamin E is a potent antioxidant that inhibits lost their initial functions to code for proteins due to membrane lipid peroxidation, modulates the metabol- the accumulation of various mutations.35 HMGA1P6 ism of arachidonic acid,41 inhibits cyclooxygenase has a well-established oncogenic action by preventing enzyme activity,42 and inhibits the release of interleukin apoptosis and enhancing cell proliferation and migra- 1b by inhibiting the 5-lipooxygenase pathway.43 tion.14,36 Overexpression of HMGA1P6 increases the Therefore, topical use of anti-allergic drugs seems 456 Laboratory Animals 53(5) strongly recommended to test a potential therapeutic Barthold SW, et al (eds) The mouse in biomedical target for the control of this disease, in order to research, 2nd ed. Burlington, MA: Academic Press, improve the refinement of animal research minimizing 2007, pp.623–717. the pain and suffering due to the devastating skin 10. Turturro A, Duffy P, Hass B, et al. Survival characteris- lesions of UD, thus relieving the distress of mice and tics and age-adjusted disease incidences in C57BL/6 mice fed a commonly used cereal-based diet modulated by improving their well-being. dietary restriction. J Gerontol A Biol Sci Med Sci 2002; 57: B379–B389. Acknowledgement 11. Perkins SN, Hursting SD, Phang JM, et al. Calorie The authors would like to thank Dr Arianna Ilsami for her restriction reduces ulcerative dermatitis and infection- valuable technical support and Dr Giuseppe Piegari for his related mortality in p53-deficient and wild-type mice. insight in statistical analysis. J Invest Dermatol 1998; 111: 292–296. 12. Blackwell BN, Bucci TJ, Hart RW, et al. Longevity, body Declaration of Conflicting Interests weight, and neoplasia in ad libitum-fed and diet-restricted C57BL6 mice fed NIH-31 open formula diet. Toxicol The author(s) declared the following potential conflicts of Pathol 1995; 23: 570–582. interest with respect to the research, authorship, and/or pub- 13. Sundberg JP and King LE. Skin and its appendages: lication of this article: On behalf of all authors the corres- normal anatomy and pathology of spontaneous, trans- ponding author states that there are no conflicts of interest. genic, and targeted mouse mutations. In: Ward JM, Mahler JF, Maronpot RR, et al. (eds) Pathology of gen- Funding etically engineered mice. Ames, IA: Iowa State University The author(s) received no financial support for the research, Press, 2000, pp.183–216. authorship, and/or publication of this article. 14. Adams SC, Garner JP, Felt SA, et al. A ‘‘pedi’’ cures all: toenail trimming and the treatment of ulcerative derma- ORCID iD titis in mice. PLoS One 2016; 11: e0144871. 15. Esposito F, De Martino M, Petti MG, et al. HMGA1 Orlando Paciello http://orcid.org/0000-0003-3091-0905 pseudogenes as candidate proto-oncogenic competitive endogenous RNAs. Oncotarget 2014; 5: 8341–8354. References 16. Committee for the update of the guide for the care and 1. Sargent JL, Koewler NJ and Diggs HE. Systematic litera- use of laboratory animals. Guide for the care and use of ture review of risk factors and treatments for ulcerative laboratory animals. 8th ed. Washington, DC: National dermatitis in C57BL/6 mice. Comp Med 2015; 65: 465–472. Academies Press, 2011. 2. Hampton AL, Hish GA, Aslam MN, et al. Progression of 17. AVMA Panel on Euthanasia. AVMA guidelines for the ulcerative dermatitis lesions in C57BL/6Crl mice and the euthanasia of animals, www.avma.org (2013, accessed development of a scoring system for dermatitis lesions. 9 May 2017). J Am Assoc Lab Anim Sci 2012; 51: 586–593. 18. Mizutani N, Sae-Wong C, Kangsanant S, et al. 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Re´sume´ La dermatite ulce´rative (DU) idiopathique est une maladie progressive et spontaneé qui touche en geńe´ral les vieilles souris C57BL/6 pre´sentant une e´tiopathogeńe`se inconnue. Pour cette e´tude, nous avons e´value´ vingt- cinq cas de DU chez des souris C57BL/6NCrl-Tg(HMGA1P6)1Pg. Des ećhantillons de peau dans du Formol et inclus en paraffine ont e´te´ soumis a` des analyses morphologiques. Une analyse immunohistochimique a e´te´ effectueé afin de caracte´riser et de quantifier les cellules inflammatoires en utilisant les anticorps CD3, CD45/B220, CD4, CD8 et l’IL-17. L’IgE lieé aux mastocytes a e´te´ e´tudieé par immunofluorescence, tandis que des ećhantillons de peau dans du se´rum et cryoconserve´s ont e´te´ pre´leve´s pour l’analyse molećulaire. Le test t de Student (double) a e´te´ effectue´ pour e´valuer les diffe´rences significatives entre les deux groupes. La peau affecteé pre´sentait d’importantes zones d’ulce´ration ainsi que des infiltrats inflammatoires diffus, se´ve`res et mixtes. Aucun changement pertinent n’a e´te´ observe´ chez les souris te´moins. L’analyse immunohistochimique a montre´ une population de leucocytes CD3 þ CD4 þ pre´dominante et moins de leucocytes CD45/B220 et de cellules IL-17 immunomarqueés et d’IgE lieé aux mastocytes. L’augmentation de l’IL-1 et du TNFa, ainsi que l’b expression de l’ARNM de l’IL-6 ont e´te´ observeés dans la peau des animaux atteints par rapport aux controˆles. L’IL-6 et le TNF se´riquea n’ont pas varie´ entre les souris atteintes et les souris te´moins. Les infiltrats inflammatoires et l’expression des cytokines e´taient cohe´rents a` la fois avec la diffe´r- enciation Th2/IgE et Th17, un mode`le typique de reáction d’hypersensibilite´ de type I. Dans l’ensemble, nos donneés sugge`rent une e´tiopathogeńe`se allergique de la DU chez les souris C57BL/6NCrl-Tg(HMGA1P6)1Pg. 458 Laboratory Animals 53(5)

Abstract Ulzerative Dermatitis (UD) ist eine idiopathische, spontane und progressive Erkrankung, die typischerweise alte C57BL/6-Maüse mit unbekannter A¨tiopathogenese betrifft. Fu¨r diese Studie haben wir fu¨nfundzwanzig Fa¨lle von UD bei C57BL/6NCrl-Tg(HMGA1P6)1Pg-Maüsen untersucht. Formalinfixierte, paraffineingebettete Hautproben wurden morphologisch begutachtet. Immunhistochemische Analyse wurde durchgefu¨hrt, um Entzu¨ndungszellen mit CD3-, CD45/B220-, CD4-, CD8- und IL-17-Antiko¨rpern zu charakterisieren und zu quantifizieren. Mastzellgebundenes IgE wurde mittels Immunfluoreszenz untersucht, wa¨hrend Serum- und kryokonservierte Hautproben fu¨r die Molekularanalyse entnommen wurden. Der t-Test (zweiseitig) wurde durchgefu¨hrt, um signifikante Unterschiede zwischen den beiden Gruppen zu beurteilen. Die betroffene Haut zeigte ausgedehnte Ulzerationen und diffuse, schwere und gemischt entzu¨ndliche Infiltrate. Bei Kontrollmaüsen wurden keine relevanten Vera¨nderungen beobachtet. Die immunhistochemische Analyse zeigte eine vorherrschende CD3 þ CD4 þ Leukozytenpopulation mit weniger CD45/B220- und IL-17- immun- markierten Zellen und mastzellgebundenem IgE. Erho¨hungen der TNFa-, IL-1b- und Il-6 mRNA-Expression wurden in der Haut der betroffenen Tiere im Vergleich zu den Kontrolltieren beobachtet. Serum TNFa und IL- 6 variierten nicht zwischen betroffenen und Kontrollmaüsen. Entzu¨ndliche Infiltrate und die Expression von Zytokinen waren sowohl mit der Th2/IgE- als auch mit der Th17-Differenzierung konsistent, ein typisches Muster einer Typ-I-U¨berempfindlichkeitsreaktion. Insgesamt legen unsere Daten eine allergisch bedingte A¨tiopathogenese von UD bei C57BL/6NCrl-Tg(HMGA1P6)1Pg nahe.

Resumen La dermatitis ulcerativa (UD) es una enfermedad idiopa´tica, espontańea y progresiva que normalmente afecta a ratones C57BL/6 mayores con etiopatogenia desconocida. Para este estudio, se han evaluado veinticinco casos de UD en ratones C57BL/6NCrl-Tg(HMGA1P6)1Pg. Se presentaron muestras de tejido fijadas en formol e incluidas en parafina para someterse a una investigacioń mofolo´gica. Se realizo´ un ana´lisis inmunohistoquı´mico para caracterizar y cuantificar las ce´lulas inflamatorias utilizando anticuerpos CD3, CD45/B220, CD4, CD8 y IL-17. Se investigo´ la IgE ligada a los mastocitos mediante inmunofluorescencia, mientras que recogieron tambień muestras de tejido crioconservados y de suero para un ana´lisis molecular. Se realizo´ una prueba T Student (de dos muestras) para evaluar las principales diferencias entre los dos grupos. Los tejidos afectados mostraron grandes a´reas de ulceracioń e infiltrados inflamatorios mezclados, agudos y difusos. No se observaron cambios relevantes en los ratones controlados. El ana´lisis inmunohistoquı´mico mostro´ una poblacioń leucocitaria CD3 þ CD4 þ predominante con menos ce´lulas inmu- noanalizadas CD45/B220 y IL-17 e IgE ligada a mastocitos. Se observo´ un aumento en la expresioń TNFa, IL- 1b e Il-6 mRNA de los tejidos de los animales afectados en comparacioń a los controles. El suero TNFa e IL-6 no variaron entre los ratones afectados y los controlados. Los infiltrados inflamatorios y la expresioń citocina fueron consistentes con la diferenciacioń Th2/IgE y Th17, un patroń comuń de un tipo de reaccioń hipersen- sible. En general, nuestros datos sugieren una etiopatogenia basada en alergia de UD en ratones C57BL/ 6NCrl-Tg(HMGA1P6)1Pg. Original Article Laboratory Animals 2019, Vol. 53(5) 459–469 ! The Author(s) 2018 Comparison of pre-emptive butorphanol or Article reuse guidelines: sagepub.com/journals- metamizole with ketamine þmedetomidine permissions DOI: 10.1177/0023677218815208 and s-ketamine þ medetomidine journals.sagepub.com/home/lan anaesthesia in improving intraoperative analgesia in mice

C Bauer1 , U Schillinger1, J Brandl1, A Meyer-Lindenberg2, A Ott3 and C Baumgartner1

Abstract In accordance with the ‘refinement’ component of the 3Rs, the primary aim of this study was to investigate and compare ketamine þ medetomidine (KM) and s-ketamine þ medetomidine (SKM) anaesthetic protocols in C57BL/6J mice (both sexes). We sought to determine whether s-ketamine could provide adequate surgical tolerance at a 50% dose relative to that of ketamine racemate and whether antagonism of medetomidine could be initiated 15 min earlier. The second aim was to investigate the potential improvement in analgesia for both anaesthetic protocols by adding butorphanol or metamizole. Analgesia was tested via the pedal withdrawal reaction (PWR) to a painful stimulus. During anaesthesia, respiratory frequency, pulse oximetry, body temperature and PWR were monitored. Among the 16 mice in each group, the PWR was lost in all the KM þ metamizole (35:56 6:07 min), KM þ butorphanol (43:45 2:14 min) and SKM þ butorphanol (24:03 5:50 min) mice, 15 of the non-premedicated KM (37:00 8:11 min) mice, and 9 of the pure SKM (20:00 4:19 min) mice; the latter group increased to 11 mice (17:16 5:10 min) with premedication of metamizole. In contrast to the racemic combination, s-ketamine at the dose used here did not lead to sufficient loss of the PWR. However, earlier partial antagonism of SKM resulted in a slightly shorter and qualitatively better recovery than later partial antagonism of SKM. The addition of metamizole or butorphanol to KM or SKM anaesthesia positively influences the analgesic quality. However, when butorphanol is added, controlled ventilation may be necessary, especially for male mice.

Keywords anaesthesia, mice, analgesia, ketamine, medetomidine, s-ketamine, metamizole, butorphanol, dipyrone

Date received: 27 November 2017; accepted: 24 October 2018

Introduction To improve animal experiments in accordance with the 1Centre of Preclinical Research, Technical University of Munich, 3R (reduction, refinement, replacement) guidelines, Germany 2Clinic for Small Animal Surgery and Reproduction, Ludwig- anaesthesia and analgesia are important factors that Maximilians-University Munich, Germany can be refined. The appropriate balanced anaesthetic 3Institute for Medical Statistics and Epidemiology, Technical regimen is determined based on the frequency and dur- University of Munich, Germany ation of the experiment and the painful stimuli. Mice are difficult animals to anaesthetize due to their high Corresponding author: 1 Caroline Bauer, Centre of Preclinical Research, Klinikum rechts risk of hypothermia and hypoglycaemia and the large der Isar, Technical University of Munich, Ismaninger Str. 22, 81675 discrepancies in doses among different strains. Beside Munich, Germany. inhalant anaesthetics only few injectable combinations Email: [email protected] 460 Laboratory Animals 53(5) can be recommended for mice. Ketamine þ a2-adreno- 15–20 weeks were included. The mice were housed ceptor agonist combinations are commonly used, but under specific-pathogen-free (SPF) conditions in large variations in analgesic depth and duration among accordance with the ‘Federation of Laboratory individuals have been reported,2–7 which makes stand- Animal Science Associations’ (FELASA) recommenda- ardization difficult and poses a risk of insufficient anal- tions.19 To reduce the number of animals used, each gesia. Therefore, we sought to improve the recovery mouse was randomly allocated to two different anaes- time and the quality of analgesia for surgical interven- thetic protocols with a wash-out period of two weeks tions. We compared ketamine þ medetomidine (KM) between experiments. In total, 48 mice (24 male, 24 and s-ketamine þ medetomidine (SKM) in C57BL/6J female) were used in this study, which equals a reduc- mice. Each protocol was also evaluated with the add- tion of experimental mice by half. Furthermore, each ition of the analgesics butorphanol and metamizole. mouse was allowed an adaptation period of at least one Butorphanol acts as an agonist mainly at j-opioid week before the first intervention. After the end of the receptors and as a partial agonist or antagonist at study and a convalescence period of at least three l-opioid receptors.8 Therefore, butorphanol has no weeks, the mice were reused in a final low-stress experi- addictive potency and is officially a non-restricted nar- ment, with permission from our approving authority. cotic drug.7,9 The pyrazolone derivate metamizole Mice were group housed (2–5 mice per cage, (syn. dipyrone) is a nonsteroidal antipyretic analgesic. 435.7 cm2 floor area) in individually ventilated cages Although the complete mechanisms of action of meta- (cage type 9, Maxi-Miser Thoren-Caging-System, mizole have not been fully clarified, metamizole has Thoren Caging Systems Inc., Pennsylvania, USA), in been shown to inhibit NDMA receptors (similar accordance with the European guidelines (2010/63/ to ketamine) and cyclooxygenase and act as an antag- EU), on special wood bedding (Select Fine, Sniff, onist of TRPA1 channels.10,11,12 S-ketamine is used at a Germany). Autoclaved standard rodent diet (Altomin 50% lower dose than racemic ketamine, as previously No. 1324SP, Lage, Germany) and water were given ad described in hamsters,13 cats14 and humans.15 libitum. Autoclaved cellulose, aspen bricks, and nestlets Therefore, we investigated whether such s-ketamine (Bioscape, Castrop-Rauxel, Germany) and a red poly- doses would also achieve an adequate anaesthetic carbonate house (Bioscape, Castrop-Rauxel, Germany) depth in mice. Furthermore, recovery was evaluated were provided for enrichment. The room temperature after antagonism of medetomidine at the earliest pos- was maintained at 22 2C and a humidity of 45–60%. sible time point. For racemic KM, the earliest antagon- Animals were maintained under a 12/12 h light/dark ism time point was after 40 min,16 without intense cycle with 30 min of dawn and dusk. ketamine-induced catalepsy. When using s-ketamine, partial antagonism can be performed even earlier, as Drugs and methods reported in hamsters.17 We additionally investigated both sexes independently, as notable sex differences Mice were not fasted before anaesthesia. After weighing related to the metabolism of these drugs have been and visual examination, either pre-emptive analgesia reported.4,6,18 was given before intraperitoneal (IP) anaesthesia or (in the groups without additional analgesia) anaesthesia was directly administered by IP injection. Material and methods Sixteen mice (8 male, 8 female) each were assigned to Ethics statement the following groups: This study was performed in accordance with the German . KM: ketamine (100 mg/kg; Narketan 100 mg/ml, Animal Welfare Act (Deutsches Tierschutzgesetz) as Ve´ toquinol GmbH, Ravensburg, Germany) and well as Directive 2010/63/EU and approved by the medetomidine (1 mg/kg) mixed in one syringe. local ethics committee of the approving authority, the . SKM: S-ketamine (50 mg/kg; Ketanest-S 25 mg/ml, district government of Upper Bavaria in Germany Pfizer Pharma PFE GmbH, Berlin, Germany) and (Gz.:55.2-1-54-2532-186-2015). The acute and cumula- medetomidine (1 mg/kg) mixed in one syringe. tive stress level was categorized as low for the current . KM þ butorphanol: butorphanol (2.5 mg/kg; study. AlvegesicÕ vet. 10 mg/ml, Selectavet, Weyarn- Holzolling, Germany) subcutaneously (SC) 30 min Animals before ketamine (100 mg/kg) and medetomidine (1 mg/kg) administration. In each group, eight male (weighing 30.4 3.0 g) and . KM þ metamizole: metamizole (150 mg/kg; metami- eight female (weighing 23.4 1.7 g) C57BL/6J mice zole 500 mg/ml, Hexal AG, Holzkirchen, Germany) (Charles River Laboratories, Sulzfeld, Germany) aged orally (PO) by licking a drop containing the Bauer et al. 461

appropriate dose directly from a 1 ml syringe. The NY, USA). All values are summarized as the mean awake mouse was therefore briefly restrained 30 min (M) standard deviation (SD). ANOVA and pairwise before ketamine (100 mg/kg) and medetomidine t-tests were used to analyse the time differences in RF, (1 mg/kg) administration. HR and BT. For SpO2, the non-parametric Kruskal- . SKM þ butorphanol: butorphanol (2.5 mg/kg) SC Wallis and Wilcoxon tests were used. For unpaired 30 min before s-ketamine (50 mg/kg) and medetomi- comparisons, such as sex differences, two-sample dine (1 mg/kg) administration. t-tests or Mann-Whitney-U-tests were performed. All . SKM þ metamizole: metamizole (150 mg/kg) PO tests were two-sided with a 5% level of significance 30 min before s-ketamine (50 mg/kg) and medetomi- (p ¼ 0.05). dine (1 mg/kg) administration. Results In the KM groups, medetomidine (Sedator 1 mg/ml Albrecht GmbH, Aulendorf, Germany) was antago- Comparative data of the anaesthetic regimens are nized after 45 min by atipamezole (5 mg/kg; Antisedan quantified as the M SD. Sex differences and differ- 5 mg/ml, Ve´ toquinol GmbH, Ravensburg, Germany) ences in the sensitive vital parameters RF and HR are SC and in the SKM groups after 30 min. shown in Tables 1–3. Only significant p-values ( < 0.05) The induction time was observed from the injection are listed. Using KM without additional analgesics, one of anaesthetics until the loss of the righting reflex. The mouse did not lose the PWR. With the use of KM plus first measurement after loss of the righting reflex was butorphanol, three of the five female mice showed a defined as time point 0. Afterwards, data were recorded short duration of excitation – defined as a stage of every 5 min until injection of the antagonist. excitement including myoclonic twitches – after loss Immediately after loss of the righting reflex, the of the righting reflex. In the SKM group without add- cornea was protected with dexpanthenol eye cream itional analgesics, the PWR could be slightly triggered (Bepanthen, Bayer Vital GmbH, Leverkusen, at several points for three male mice. Four mice did not Germany). The recovery time was defined from the show a loss of this reaction at any time during anaes- injection of the antagonist until the righting reflex was thesia. One of the three male mice showed a short regained. Immediately before injecting the antagonist, period of catalepsy – defined as stage of immobilization each mouse was administered a pre-warmed infusion combined with a noticeably increased muscle tone – SC (0.2 ml Ringer’s solution þ 0.1 ml 20% glucose). after loss of the righting reflex. The PWR was triggered Monitoring of anaesthesia was performed by means slightly at several points for three mice (one male, two of the respiratory frequency (RF; respiration counted by female) using SKM þ metamizole. Two mice did not the investigator), heart rate (HR), peripheral oxygen sat- show a loss of this reaction at any time during anaes- uration (SpO2) and rectal body temperature (BT) via a thesia. Five female mice showed a short period of exci- high-speed pulse oximeter including a temperature probe tation after loss of the righting reflex. In the (‘PhysioSuite’ including ‘Thermostat’, Kent Scientific SKM þ butorphanol group, four mice (three male, Corp., USA distributed by EMKA Technologies, Paris, one female) showed a short period of excitation after, France). Additionally, the pedal withdrawal reaction and two female mice before loss of the righting reflex. (PWR; pinching the toes with atraumatic forceps) was Data are not shown for SpO2 and BT, which were evaluated (positive reaction: mouse attempt to withdraw influenced by supplemental oxygen and heat. As the limb; negative reaction: mouse did not react to toe expected, a massive decrease in both parameters was pinch). Physiological BT was maintained at 38–39Cbya observed at only time point 0 (directly after induction) heating pad (Horn GmbH, Gottmadingen, Germany), before external supplementation was initiated. and 100% oxygen was supplied via a nose mask. However, the data quickly stabilized thereafter within 7 Surgical tolerance was defined as the period when a physiological range of 37.5–38.5 C. SpO2 revealed the PWR to a painful stimulus was lost without any sufficient spontaneous breathing ( > 95 vol%) in all significant increase in HR or RF. In this study, the groups. In only male mice administered KM þ only painful stimulus was a toe pinch with atraumatic butorphanol, SpO2 was decreased to 93 6 vol% at forceps to test the PWR. 40 min; additionally, rattling breathing indicative of increased bronchial secretion was observed in some of Statistical analysis these mice. The study was classified as an explorative study and the Discussion sample size was set at n ¼ 8 in accordance to Arras et al.2 The data were analysed using Excel and IBM A refinement of anaesthetic protocols for experimental SPSS Statistics, version 24 (IBM Corp., Armonk, mouse studies according to the 3Rs was aimed in the 462

Table 1. Comparison of anaesthetic stages and pedal withdrawal reaction (PWR) among groups (in mean standard deviation).

KM þ SKM þ KM þ SKM þ KM SKM metamizole metamizole butorphanol butorphanol

Number of mice with loss of the PWR 15 of 16 9 of 16 16 of 16 11 of 16 16 of 16 16 of 16 Duration of the loss of the PWR (min) 37:00 8:11 20:00 4:19 35:94 6:07 17:16 5:10 43:45 2:14 24:03 5:50 Time point when more than 50% of mice TP 10 TP 25 TP 10 TP 20 TP 5 TP 10 ( > eight mice) lost the PWR earliest Induction time (min) < 3:14 4:45 2:43 1:22 1:36 0:33 1:45 0:45 1:07 0:24 1:35 0:49 , 1:13 0:23 1:57 1:02 1:18 0:42 2:02 1:10 1:23 0:31 1:15 0:33 Number of mice 2 < 3 < 1 < 1 < showing catalepsya 1 , Number of mice 1 , 2 < 3 , 5 , 1 < 3 < showing excitationsa 3 , 5 , 3 , Recovery time (min) < 5:29 2:56 3:10 1:44 7:25 3:57 3:17 1:01 17:04 3:22 4:41 2:36 , 5:42 1:54 4:36 2:24 8:23 6:05 2:59 1:03 13:17 4:14 5:33 2:05 Number of mice showing 2 < 6 < 8 < 6 < 6 < 8 < moderate ataxia 1 , 5 , 8 , 8 , 6 , 8 , Duration of moderate ataxia (min) 26:08 17:34 19:38 12:21 33:18 15:29 26:25 11:50 42:05 18:01 24:11 12:25 Number of mice 1 < 1 , 7 < 6 < 6 < 8 < showing high-grade ataxia 3 , 8 , 7, 8 , 8 , Duration of high-grade ataxia (min) 28:45 23:13 5:00 28:44 19:05 15:41 6:26 65:21 26:42 43:33 26:49

< male, , female. 53(5) Animals Laboratory KM: ketamine þ medetomidine, SKM: s-ketamine þ medetomidine. Loss of the PWR: no pedal withdrawal reaction to a painful stimulus. Time points (TP) started with TP 0 ¼ loss of the righting reflex; TP were measured every 5 min. aIncluding catalepsy/excitations before and after the loss of the righting reflex. ae tal. et Bauer

Table 2. Comparison of heart rate during anaesthesia among groups and sex (in mean standard deviation).

Anaesthesia groups TP 0 TP 5 TP 10 TP15 TP 20 TP 25 TP 30 TP 35 TP 40 TP 45

KM 387 83 370 78 340 56 345 53 354 52 351 51 346 51 346 51 338 53 336 53 male 371 79 383 80 357 64 372 60 391 42 385 42 375 52 375 52 364 59 360 62 female 404 89 358 80 324 45 318 26 317 30 317 33 316 30 317 32 313 33 313 33 SKM 308 67 266 46 278 48 293 46 303 47 312 42 308 40 Statistical significance p ¼ 0.005a p ¼ 0.046a male 307 29 282 49 294 44 305 35 323 32 329 25 327 22 female 310 102 250 40 262 48 281 55 284 54 295 50 290 46 KM þ Metamizole 301 79 329 88 348 66 355 61 356 61 362 64 356 62 362 59 358 56 359 51 Statistical significance p ¼ 0.039b male 293 71 340 82 372 73 372 73 363 76 374 78 367 75 369 69 364 66 358 65 female 310 94 318 97 325 52 338 44 348 47 351 49 346 47 355 51 352 46 360 38 SKM þ Metamizole 258 42 259 43 278 46 296 57 305 50 312 53 321 54 Statistical significance p ¼ 0.012a p ¼ 0.000a p ¼ 0.007a p ¼ 0.000b male 269 47 265 44 291 53 311 68 322 59 327 57 340 57 female 249 39 254 44 266 39 283 46 291 38 298 48 305 49 KM þ Butorphanol 249 65 330 55 357 45 364 45 362 43 354 40 347 38 344 35 338 31 340 30 Statistical significance p ¼ 0.002a p ¼ 0.000b male 237 34 306 43 371 51 374 52 372 59 371 50 359 48 358 40 355 25 359 23 female 262 87 355 57 342 35 354 37 352 18 338 17 335 20 330 26 320 28 324 26 SKM þ Butorphanol 209 27 248 48 265 51 286 51 304 47 312 44 317 45 Statistical significance p ¼ 0.00a p ¼ 0.0014a p ¼ 0.000a p ¼ 0.001a p ¼ 0.000b male 213 17 259 54 287 57 309 55 329 51 328 49 331 51 female 206 35 237 43 244 35 264 37 279 28 297 33 303 35

Table 3. Comparison of respiratory frequency during anaesthesia among groups and sex (in mean standard deviation). anaesthesia groups TP 5 TP10 TP15 TP 20 TP 25 TP 30 TP 35 TP 40 TP 45

KM 150 23 158 22 162 21 163 20 166 22 168 19 167 19 171 18 172 19 Statistical significance p ¼ 0.011a p ¼ 0.014a p ¼ 0.00b male 162 17 167 20 169 17 170 14 173 18 173 15 171 17 175 19 173 18 female 139 24 148 21 155 23 156 23 159 25 163 22 162 20 167 17 170 21 SKM 138 20 144 15 150 17 155 18 155 15 156 13 Statistical significance p ¼ 0.036a p ¼ 0.024a p ¼ 0.001b male 149 15 154 10 158 12 164 6 162 12 159 10 female 128 20 134 14 142 18 145 20 149 16 152 15 KM þ Metamizole 163 24 168 23 169 20 172 23 175 25 177 21 178 19 175 18 183 19 Statistical significance p ¼ 0.024a p ¼ 0.007b male 165 28 171 22 168 21 176 20 178 23 178 22 177 17 176 18 184 20 female 162 22 165 25 171 20 167 26 173 29 177 22 179 22 175 19 181 19 SKM þ Metamizole 156 22 158 22 158 20 160 20 162 15 160 18 male 157 22 158 28 155 25 161 21 162 16 158 24 female 156 23 159 16 162 17 160 19 162 16 163 13 KM þ Butorphanol 132 14 130 14 130 13 132 12 133 9 133 13 135 10 135 11 136 11 male 129 11 131 13 129 13 131 14 131 10 131 12 133 11 133 13 135 13 female 134 16 130 15 131 14 134 10 135 10 134 14 137 9 138 9 138 10 SKM þ Butorphanol 123 20 127 19 129 16 128 17 130 17 135 16 Statistical significance p ¼ 0.01b male 124 22 130 22 132 19 130 20 135 18 139 20 female 122 20 124 17 126 12 125 13 126 15 132 13

KM: ketamine þ medetomidine, SKM: s-ketamine þ medetomidine; TP: time point. aSignificant difference between current and former time points. bSignificant difference between time point 0 and end time point. current study by using s-ketamine at half of the dose of was not satisfying since only nine out of 16 mice racemic ketamine in combination with medetomidine, (three male, six female) lost the PWR for while inducing an adequate surgical tolerance level 20:00 4:19 min. Sex differences due to drug dosages as successfully reported in hamsters,13 cats14 and in another mouse strain have previously been reported humans.15 Furthermore, anaesthesia may be better con- by Cruz et al.,4 whom, in contrast to our findings, trolled by earlier antagonism of medetomidine to avoid observed that female Swiss Webster mice needed 55% a potential ketamine hangover with intense catalepsy.16 more ketamine than male mice. Catalepsy during induc- Additionally, analgesia for surgical procedures may be tion occurred in only a few male mice and one female improved and prolonged by the addition of metamizole mouse, whereas excitations were observed more often in or butorphanol to basic KM or SKM anaesthesia. female mice. Ataxia was observed in all groups after To test nociception during anaesthesia, we used the the righting reflex was regained, as also reported by PWR as a reliable indicator of reaching surgical toler- the literature,21,22 and lasted longer in the KM group ance and sufficient analgesia in accordance with the than in the SKM group, which was already known in studies of Arras et al.2 and Buitrago et al.20 hamsters13 and mice.21 Ataxia lasted for at most 2 h, and The dose of ketamine (100 mg/kg) and medetomidine after this time, the mice showed normal locomotion and (1 mg/kg) was based on that used by Arras et al.2 In behaviour. This finding concurs with those of a study by contrast to that of SKM (50 mg/kg), the chosen dose Hja´ lmarsdottir.22 High-grade ataxia was more prevalent of KM was sufficient to achieve a loss of PWR in in female mice. This could have been caused by the keta- nearly all of the C57BL/6J mice for a duration mine which induces catalepsy by increasing the tonus of (37:00 8:11 min) sufficient for surgical procedures, skeletal muscles,7 and because of the persistent activity although results of KM anaesthesia were not sufficient of the muscles due to increased action in some subcor- in other studies.2,3,6,20 In contrast, the tested SKM dose tical regions,10 spontaneous limb movements can occur.7 Bauer et al. 465

This finding is also in accordance with that from vasoconstriction results in an increase in blood pressure Cesarovic et al.,23 in which only s-ketamine was given due to increased peripheral vessel resistance.7 As blood as premedication, and all mice exhibited ataxia and pressure was not monitored in the current study, we can tremor during induction. Furthermore, mice are a only assume the simultaneous regulation of HR and highly reflex-oriented species.22 Thus, reflex activity can blood pressure. persist while the dose of narcotics cannot be further Overall, males showed higher RF and HR than increased without risk of death.24 Caused by the partial females, except the female mice of the KM, SKM, antagonization of medetomidine, ataxia may be attri- KM þ metamizole and KM þ butorphanol groups, bute to the described ketamine effect. In the s-ketamine which initially had a higher HR at time point 0 than protocols, the lower dose of s-ketamine may be respon- the male mice. This may be due to stress, which affects sible for the shorter duration of ataxia. HR29 and RF. As conscious female mice of these Sex differences were also observed during induction groups showed a higher level of activity and defence with SKM and KM, in which unconsciousness and loss reactions than male mice, the higher initial HR may of the PWR were achieved earlier in females than in be caused by their higher stress level during handling males. This result may be caused by the higher percent- for drug administration. Hence, the induction time was age of fat in male mice than in female mice, as more very fast, and the adaptation of HR to anaesthesia in lipophilic ketamine25 accumulates in fat,26 which may female mice may have taken longer than that in male lead to a delayed effect. Before additional heat supply, mice. In accordance with the study by Xing et al.,30 the the male mice of the KM group had a higher BT than higher HR in male C57BL/6J mice after administration the female mice, as also reported by Cruz et al.4 This of anaesthesia may be influenced by their genetic back- difference may be due to the lower percentage of muscle ground. Flandre et al.31 previously reported a higher in females than in males.27 Within the SKM group, only RF in male mice than in female mice, which may three of the male mice, compared to six of the female have been caused by differences in hormones.32 When mice, showed loss of the PWR over a certain time. As using SKM, inspiratory breathing was deeper and more s-ketamine is the right-handed enantiomer of racemic effective in females than in males, as females regained ketamine,15 this sex difference may be caused by possible physiological SpO2 immediately after receiving an differences in the enzymatic system of hepatic metabol- oxygen supply, whereas males regained physiological ism.26 The slight, continuous increase in RF over the SpO2 only after 15 min, despite their higher RF. course of anaesthesia, especially in the KM, SKM, Additionally, according to Voipio et al.,6 females take KM þ metamizole and SKM þ butorphanol groups, longer to regain the righting reflex during recovery than may be due to increased BT, pain, insufficient anaes- males, which may be caused by different hepatic metab- thetic level, hypoxia or hypercapnia. Due to the applica- olism of (s-)ketamine between males and females.26 tion of an external heat supply, the BT slowly increased For ketamine þ a2-agonist combinations, the depth during the course of anaesthesia to a physiological level and duration of surgical tolerance vary widely among but not to hyperthermia. We can exclude pain and individuals.2,3,5,6,20,21 Therefore, we investigated two an insufficient anaesthesia level as reasons for additional analgesics that are commonly used in veter- the respiratory depression, as the mice simultaneously inary practice and are useful for treating moderate exhibited loss of the PWR. Hypoxia was not observed pain. Depending on the painful stimulus of the experi- by pulse oximetry, except in some males of the ment, more potent analgesics could be necessary. KM þ butorphanol group, in which light hypoxia Butorphanol, an opiate agonist-antagonist,7 was rec- (SpO2 < 95 vol%) was detected. Regarding hypercapnia, ommended for use combined with dexmedetomidi- spontaneous respiration during anaesthesia may have ne þ tiletamine þ zolazepam in mice by Cagle et al.33 led to sufficient but reduced gas exchange, as breathing and combined with medetomidine þ midazolam by became shallower. A compensatory reaction would have Kawai et al.34 As shown in other species35,36 and in increased the RF. mice,37,38 metamizole is a potent analgesic and can The considerable decrease in HR in the SKM and reduce the required dose of other anaesthetics when KM groups followed by a continuous increase in HR administered in combination. The doses of butorphanol after 15 min of anaesthesia may be caused by the phen- and metamizole were chosen in accordance with cyclidines and a2-agonists. Ketamine likely activates Erhardt et al.7 the central sympatho-adrenergic system and depresses With the exception of the SKM þ metamizole group, the reuptake of noradrenaline at the synaptic junction. the PWR was lost in all groups with additional admin- Due to these effects, stimulation of the cardiovascular istration of analgesia. However, compared to pure system follows, as indicated by tachycardia and hyper- SKM treatment, metamizole premedication was able tension.7 Medetomidine typically causes bradycardia, to increase the percentage of mice that lost the PWR and within the first few minutes, peripheral from 56 to 69%. Anaesthesia was induced more quickly 466 Laboratory Animals 53(5) with premedication in male mice as well as in female premedicated with butorphanol. Ketamine induces sal- mice of the SKM þ butorphanol group than without ivary and bronchial secretion,10,44 which appears to be premedication (see Table 1), which can be explained intensified by the addition of butorphanol, especially in by the sedative effect of butorphanol7 and by the antag- males. Whether premedication with atropine, as recom- onistic effect of metamizole on NMDA receptors of the mended by Flecknell,44 is able to reduce this bronchial central nervous system.11,39 The combination of secretion needs to be examined in further studies. KM þ butorphanol induced the best analgesic effect. Additionally, the male mice of the KM þ butorphanol However, the addition of butorphanol to KM led to group required the longest recovery period. As male more side effects, especially short-term mild to moder- mice have a higher percentage of fat, the extended recov- ate excitation. Opioids are able to reduce the threshold ery may be caused by the lipophilic property of butor- of excitation in the central nervous system as they are phanol,46 which may possibly allow butorphanol to weak partial m-agonists.7,40 In contrast, all four preme- accumulate in the body. dicated groups showed less catalepsy than the groups The noticeable difference in sex in all premedicated without premedication, as both butorphanol41 and groups has been previously shown for basic anaesthe- metamizole may reduce the high skeletal muscle tonus sia, as the mean HR in male mice is consistently higher caused by (s-)ketamine. As metamizole is known to than that in female mice. Xing et al.30 also reported a induce spasmolytic action in smooth muscles, sup- higher HR in male C57BL/6J mice than in female mice. posedly by inhibiting the release of intracellular A key reason for this sex difference, aside from an age Ca2þ,7,42 it may also influence skeletal muscle when difference,30 is the contrasting hormone activity, which administered in combination with a2-agonists, as not affects the cardiovascular system by modulating ven- all mechanisms of action have been identified thus far. tricular repolarization.47 In the premedicated KM groups, both metamizole and In summary, our aims were not completely achieved, especially butorphanol clearly prolonged the time to as the PWR was not lost in all mice under the s-ketamine regaining the righting reflex after antagonism, which protocol, which has also been shown in dogs.48 is caused by the slight sedative effect of butorphanol33 Therefore, more detailed dose-evaluation studies on the that was unaffected by atipamezole antagonism9 and by SKM combination are necessary. However, earlier the dose-dependent sedative effect of metamizole.43 The antagonism of medetomidine in combination with mean RF, HR and SpO2 were lower in the butorphanol s-ketamine resulted in a qualitatively better and shorter groups directly after induction than in the metamizole recovery. Improvements in analgesia were observed fol- groups. In contrast to basic KM and SKM, all groups lowing the use of (s-)ketamine þ medetomidine protocols that received premedication started with a markedly with the addition of either butorphanol or metamizole. lower HR at time point 0, but the HR increased signifi- The addition of butorphanol to the basic anaesthetic cantly during the anaesthetic period (p-values are protocols markedly increased the mean duration for shown in Table 3). The opioid analgesic butorphanol which the PWR was absent. The addition of metamizole produces a sedative effect on the central nervous system or butorphanol to SKM increased the number of mice and depresses breathing by affecting m-opioid recep- that exhibited a loss of the PWR. Premedication, espe- tors25,40 and circulation (blood pressure and HR). cially by pre-emptively administered butorphanol, Metamizole is known to decrease blood pressure after allowed a notably shorter anaesthesia induction time fast intravenous injection.7 In this study, metamizole than that without premedication, especially in males, was given orally. As no primary decrease in HR was and decreased ketamine-based catalepsy. However, the observed directly after induction in the metamizole excitation rate during induction increased when using groups, orally administered metamizole does not seem the KM-based protocols. A disadvantage of adding to notably influence the cardiovascular system. This butorphanol to KM was the development of insufficient observation may be due to the influence of stomach respiration and massive bronchial secretion in male mice content on the onset of oral medication,45 as rodents after 40 min. Therefore, equipment for intubation and do not need to be fasted prior to anaesthetic induction.44 mechanical ventilation should be available when using Supplementation of 100% oxygen under spontaneous this combination. Regarding recovery, butorphanol had breathing was sufficient in all mice except male mice of a longer lasting sedative effect than metamizole, and the KM þ butorphanol group beginning at time point ataxia was more pronounced. A dose reduction should 40, at which time these mice reached only 93 6vol%, be considered when using butorphanol in this mouse and some exhibited the rattling breathing characteristic strain. No disadvantage was observed following the of increased bronchial secretion. Massive bronchial use of metamizole, and pre-operative application of secretion during recovery was observed in 10 out of 16 this analgesic in combination with KM and SKM is male mice and in half of the female mice of both groups therefore highly recommended. Bauer et al. 467

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Re´sume´ Conforme´ment a` la composante de « raffinement » des 3R, le principal objectif de cette e´tude e´tait d’e´tudier et de comparer les protocoles anesthe´siques associant d’une part la ke´tamine þ me´de´tomidine (KM) et d’autre part la s-ke´tamine þ me´de´tomidine (SKM) chez les souris C57BL/6J (des deux sexes). Nous avons cherche´ a` de´terminer si la s-ke´tamine pourrait fournir une tole´rance chirurgicale ade´quate a` une dose de 50% par rapport a` celle de la ke´tamine race´mique et si l’antagonisme de la me´de´tomidine pouvait eˆtre initie´ 15 min plus toˆt. Le deuxie`me objectif e´tait d’e´tudier l’ame´lioration potentielle de l’analge´sie dans le cas des deux protocoles anesthe´siques en ajoutant du butorphanol ou du me´tamizole. L’analge´sie e´tait testeé via le re´flexe de retrait de la patte (RRP) a` un stimulus douloureux. Au cours de l’anesthe´sie, l’oxyme´trie du pouls, la fre´quence respiratoire, la tempe´rature corporelle et le RRP ont e´te´ surveille´s. Parmi les 16 souris de chaque groupe, toutes celles du groupe KM þ me´tamizole (35:56 6:07 min), du groupe KM þ butorphanol (43:45 2:14 min) et du groupe SKM þ butorphanol (24:03 5:50 min) ont perdu ce re´flexe, ainsi que 15 des Bauer et al. 469 souris n’ayant pas fait l’objet d’une pre´me´dication KM (37:00 8:11 min) et 9 des souris du groupe SKM pur (20:00 4:19 min); ce dernier groupe est passe´ a` 11 souris (17:16 5:10 min) avec une pre´me´dication au me´tamizole. Contrairement a` la combinaison race´mique, la s-ke´tamine a` la dose utiliseé ici n’a pas conduit une perte suffisante du re´flexe. Toutefois, l’antagonisme partiel de SKM plus prećoce a donne´ lieu a` une rećupe´ration un peu plus courte et qualitativement meilleure que l’antagonisme partiel plus tardif de SKM. L’ajout de me´tamizole ou de butorphanol a` l’ane´sthe´sie par KM ou SKM influence positivement la qualite´ de l’analge´sique. Cependant, lorsque le butorphanol est ajoute´, une ventilation controˆleé peut s’ave´rer nećes- saire, en particulier pour les souris maˆles.

Abstract Im Einklang mit der ‘‘Verbesserungskomponente’’ des 3R-Prinzips (Verbesserung, Verringerung, Vermeidung) war das Hauptziel dieser Studie die Untersuchung und der Vergleich von Ketamin þ Medetomidin (KM) und S-Ketamin þ Medetomidin (SKM) basierten Ana¨sthesieprotokollen bei C57BL/6J- Maüsen (beide Geschlechter). Wir wollten ermitteln, ob S-Ketamin eine ausreichende chirurgische Toleranz bei einer Dosis von 50% im Vergleich zu der des Ketamin-Racemat bieten wu¨rde und ob der Medetomidin-Anteil 15 Min fru¨her antagonisiert werden kann. Das zweite Ziel war es, eine mo¨gliche Verbesserung der Analgesie fu¨r beide Ana¨sthesieprotokolle durch Zugabe von Butorphanol oder Metamizol zu untersuchen. Die Analgesie wurde u¨ber die Fubru¨ckziehreaktion (PWR) auf einen schmerzhaften Reiz getestet. Wa¨hrend der Ana¨sthesie wurden Atemfrequenz, Pulsoximetrie, Ko¨rpertemperatur und PWR u¨ber- wacht. Von den 16 Maüsen in jeder Gruppe zeigte sich ein Verlust der PWR bei allen Maüsen der KM þ Metamizol (35:56 6:07 Min)-, KM þ Butorphanol (43:45 2:14 Min)- und SKM þ Butorphanol (24:03 5:50 Min)-Gruppen, bei 15 der nicht-pra¨medizierten KM (37:00 8:11 Min)-Gruppe und bei 9 der reinen SKM (20:00 4:19 Min)-Gruppe. Diese letzte Gruppe wurde auf 11 Maüse (17:16 5:10 Min) mit Pra¨medikation von Metamizol erho¨ht. Im Gegensatz zur racemischen Kombination fu¨hrte S-Ketamin in der hier verwendeten Dosis nicht zu einem ausreichenden PWR-Verlust. Eine fru¨here Antagonisierung des Medetomidin-Anteils der SKM-Gruppe fu¨hrte jedoch zu einer etwas schnelleren und qualitativ besseren Erholung. Die Zugabe von Metamizol oder Butorphanol zu KM- oder SKM-Narkosen beeinflusst die analge- tische Wirkung positiv. Bei Butorphanolzugabe ist u. U. jedoch eine kontrollierte Beatmung erforderlich, insbesondere bei ma¨nnlichen Maüsen.

Resumen De acuerdo con el componente de «refinamiento» de las 3R, el objetivo principal de este estudio fue investigar y comparar los protocolos de anestesia con ketamina þ medetomidina (KM) y s-ketamina þ medetomidina (SKM) en ratones C57BL/6J (de ambos sexos). Querıámos determinar si la s-ketamina podıá ofrecer una tolerancia quiru´rgica adecuada a una dosis del 50% relativa a la de racemato de ketamina y si el antagano- nismo de medetomidina podıá iniciarse 15 min antes. El segundo objetivo era investigar la mejora potencial en la analgesia para ambos protocolos aneste´sicos anãdiendo butorfanol o metamizol. La analgesia fue probada a trave´s de la reaccioń de retiro pedal (PWR) ante un estı´mulo doloroso. Durante la anestesia, se controlo´ la frecuencia respiratoria, la oximetrıá del pulso, la temperatura corporal y la PWR. Entre los 16 ratones de cada grupo, la PWR se perio´ en todos los ratones con KM þ metamizol (35:56 6:07 min), KM þ butorfanol (43:45 2:14 min) y SKM þ butorfanol (24:03 5:50 min), 15 de los ratones no premedicados KM (37:00 8:11 min) y 9 de los ratones puros SKM (20:00 4:19 min); el u´ltimo grupo aumento´ a 11 ratones (17:16 5:10 min) con premedicacioń de metamizol. En contraste con la combinacioń race´mica, s-ketamina a la dosis utilizada aquı´ no llevo´ a una pe´rdida suficiente de la PWR. Sin embargo, un antagonismo parcial anterior de SKM resulto´ en una recuperacioń ligeramente inferior y cualitativamente mejor que el antagonismo parcial posterior de SKM. La adicioń de metamizol o butorfanol a la anestesia KM o SKM influye positivamente en la calidad analge´sica. No obstante, cuando se anãde butorfanol, puede que sea necesaria una ventilacioń controlada, especialmente para los ratones machos. Original Article Laboratory Animals 2019, Vol. 53(5) 470–477 ! The Author(s) 2018 Acinetobacter species in laboratory Article reuse guidelines: sagepub.com/journals- mice: species survey and antimicrobial permissions DOI: 10.1177/0023677218818598 resistance journals.sagepub.com/home/lan

Laurentiu Benga1 , Andrea T. Feßler2, W. Peter M. Benten1, Eva Engelhardt1, Karl Ko¨hrer3, Stefan Schwarz2 and Martin Sager1

Abstract The extra-hospital epidemiology of Acinetobacter infections is a subject of debate. In recent years, the prevalence of animal multidrug-resistant Acinetobacter infections has increased considerably. The goal of the present study was to specify Acinetobacter species isolated from laboratory mice and to test them for their antimicrobial susceptibility. During routine microbiological monitoring of laboratory mice, 12 Acinetobacter spp. were isolated. By means of 16S rRNA and rpoB gene sequencing, seven of the isolates were identified as Acinetobacter radioresistens, three isolates belonged to Acinetobacter genomospecies 14BJ, one isolate was classified as Acinetobacter pitii and one as Acinetobacter sp. ANC 4051. The distribution of the minimal inhibitory concentration (MIC) values was uniform for 21 of the 23 antimicrobial agents tested, whereas a broad MIC distribution was recorded for tulathromycin and streptomycin. The MIC values recorded were low for the majority of the antibiotics tested. Nevertheless, very high MIC values, which will probably render a therapeutic approach using these substances unsuccessful, were recorded for florfenicol, tiamulin, tilmicosin and cephalothin in most of the isolates. In conclusion, we document colonization of laboratory mice with different Acinetobacter species, displaying similar antibiotic susceptibility profiles, with possible implications in the Acinetobacter epidemiology as well as in the husbandry and experimentation of the colonized animals.

Keywords health monitoring, Acinetobacter, laboratory mice, animals, antibiotic resistance

Date received: 24 April 2018; accepted: 14 November 2018

Acinetobacter species are regarded as a major infectious Acinetobacter species in laboratory rodents is known, problem in human medicine, particularly in immuno- to date there is only one study reporting the isolation compromised patients.1 The Acinetobacter strains and phenotypical identiﬁcation of Acinetobacter strains involved in nosocomial infections are frequently multi- using the Vitek-2 system.11 Animal Acinetobacter drug-resistant.2 The extra-hospital epidemiology of human Acinetobacter infections is a subject of 1Central Unit for Animal Research and Animal Welfare Affairs, debate.3 Acinetobacter species are ubiquitous within University Hospital, Heinrich - Heine - University, Du¨sseldorf, the environment and can be found in soil, water, on Germany 2Institute of Microbiology and Epizootics, Centre for Infection surfaces or in food products.4,5 Moreover, they seem 6,7 Medicine, Department of Veterinary Medicine, Freie Universita¨t to persist for long periods of time in the environment. Berlin, Berlin, Germany During recent years, the prevalence of animal multi- 3Biological and Medical Research Center (BMFZ), Heinrich - drug-resistant Acinetobacter infections has increased Heine - University, Du¨sseldorf, Germany considerably. Acinetobacter isolates have been collected Corresponding author: over time mainly from cats and dogs, but also from Laurentiu Benga, Geb. 22.22 Universita¨tsklinikum Du¨sseldorf horses, donkeys, cattle, goats, birds and even from rab- Universita¨tsstr. 1, 40225 Du¨sseldorf, Germany. 3,8–10 bits and guinea pigs. Although the presence of Email: [email protected] Benga et al. 471 isolates not only present concerns regarding infections of

humans that are in contact with animal carriers, but can rpoB also produce serious animal diseases ranging from localized infections to septicemia.10,12 Concerns for animal welfare or pathology, as well as for experimentation and the husbandry of Acinetobacter spp. infected groups of animals are aspects that should not be neglected. The GenBank access no 16S rDNA / HG917870 / LT796224 FELASA recommendations for health monitoring imply examination for infectious agents other than those listed in the recommendations, whenever considered relevant for a microbiological unit, thus granting ﬂexibility in the design of the microbiological surveillance programs of a facility.13 The goal of the present study was to specify Acinetobacter spp. isolates obtained from laboratory mice and to investigate them for their (99 / DQ207489.1) LT796211 / LT796220 (99 / DQ207489.1) LT796213 / LT796222 (99 / DQ207489.1) HG917871 / LT796223 (98 / EU477112.2) HG917872 / LT796225 (98 / EU477112.2) LT796216 / LT796228 (99 / EU477112.2) LT796218 / LT796230 antimicrobial susceptibility. (99 / EU477112.2) LT796219 / LT796231

Materials and Methods (99 / JQ838184.1) sp. ANC 4051 (99 / KJ956432.1) LT796212 / LT796221 genomosp. 14BJ (97 / EU477147.2) LT796214 / LT796226 genomosp. 14BJ (97 / EU477147.2) LT796215 / LT796227 Bacterial isolation genomosp. 14BJ (97 / EU477147.2) LT796217 / LT796229 The bacterial strains included in this study (Table 1) were isolated during the routine microbiological monitoring program of laboratory mice from the Animal Research identification (% similarity closest species / Facility of the Heinrich-Heine-University Du¨sseldorf, rpoB GenBank no. of the sequence) Acinetobacter radioresistens Acinetobacter Acinetobacter radioresistens Acinetobacter radioresistens Acinetobacter pitii Acinetobacter radioresistens Acinetobacter Acinetobacter Acinetobacter radioresistens Acinetobacter Acinetobacter radioresistens Acinetobacter radioresistens based on FELASA recommendations.13,14 The health monitoring program was approved by the competent supervisory authority as part of the breeding program. All procedures were performed in accordance with the German legislation for the care and use of laboratory animals. The Animal Research Facility is a breeding and experimental facility, housing approximately 20,000 mice, including more than 500 genetically engineered strains, mainly on C57BL6/J and BALB/c backgrounds. (100 / BAGY01000082) (99.6 / BAGY01000082) (99.8 / BAGY01000082) (99.8 / BAGY01000082) (99.8 / BAGY01000082) (99.9 / BAGY01000082) The animals are housed either in open or in individually (99.8 / BAGY01000082) (99.3 / AIEC01000082) ventilated cages in several microbiological units with dif- (97.4 / APOS01000028) ferent microbiological status. The mice are kept on wood (97.2 / APOS01000028) fiber or cellulose bedding and recieve food and water ad (97.7 / APPX01000010) libidum. Colony health is monitored routinely by a com- (99.8 / APQP01000001) bined statistically valid sampling strategy of BALB/c bed- species isolates. ding sentinels and of resident mice. Additionally, all animals from all housing areas that appear to be sick are examined. Overall, approximately 1000 mice are examined per year in the entire facility. Acinetobacter 16S rRNA identification (%GenBank similarity no. closest of species the / type strain sequence) Acinetobacter radioresistens Acinetobacter calcoaceticus Acinetobacter radioresistens Acinetobacter radioresistens Acinetobacter pitii Acinetobacter radioresistens Acinetobacter junii Acinetobacter guillouiae Acinetobacter radioresistens Acinetobacter guillouiae Acinetobacter radioresistens For health monitoring, the mice were euthanized by Acinetobacter radioresistens the CO2 method, in accordance with the directive 2010/ 63/EU, and cecum and/or feces and skin samples as well as swabs of the nasal cavities, oro-pharynx, and genital mucosa were cultured on Columbia blood and MacConkey agar plates (bioMe´rieux, Nu¨rtingen, Germany) for approximately 48 h at 37C in aerobic Source of isolation and anaerobic conditions. This is to examine the sam- Identification of mice ples for the presence of the bacteria listed in Table 3 of the FELASA recommendations that are able to grow 13 Table 1. Isolate 1300/07 caecum 1195/08 feces mouse 55/11 oro-pharynx 72/11 oro-pharynx 272/11 oro-pharynx 764/11 caecum 658/141234/14 oro-pharynx cecum 1258/14a cecum 1258/14b cecum 1301/14 cecum on the aforementioned agar media. 1302/14 skin 472 Laboratory Animals 53(5) Bacterial species identification trimethoprim-sulfamethoxazole). The test panel was Single isolated colonies were sub-cultured and tested based on the microtiter plates used for Gram-negative for purity and then subjected to phenotypic identifica- bacteria in the German National Resistance tion by colony morphology, ability to grow on special Monitoring program of veterinary pathogens GERM- media, cell morphology after Gram staining, spore Vet (https://www.bvl.bund.de/EN/09_Laboratories/ forming ability, respiratory type, motility, oxidase and 01_Tasks/03_national_resistance_monitoring/resis- catalase tests. The assignment to the genus tance_monitoring_node.html). For antimicrobial sus- Acinetobacter was based presumptively on the growth ceptibility testing, fresh overnight cultures on blood on MacConkey agar, Gram staining, a negative oxidase agar plates were used, and a density of McFarland 0. test, and the results of the API 20 NE (bioMe´rieux, 5 was adjusted in 0.85 % saline. The inoculum was Marcy l’E´toile, France) test. Further identification to prepared by adding 5 mL of the bacterial suspension species level was performed by partial sequencing of the per 1 mL of cation-adjusted Mueller-Hinton broth. 16S rRNA and rpoB genes. DNA for PCR amplifica- From this suspension, 50 mL was then pipetted into tion of the two genes was extracted from 24-h-old each well of the microtiter plate and the results were blood-agar-grown cultures as described previously.15 read after 20–24 h of incubation at 35C Æ 2C.20 The Amplification of the 16S rDNA and analysis of the Escherichia coli reference strain ATCCÕ 25922 served sequences was performed as described previously.16 A as quality control. 16S rDNA sequence similarity of 97% with that of an Acinetobacter species type strain was considered as sufficient for identification at genus level.17,18, and a pre- Results requisite for further species identification based on rpoB Identification of Acinetobacter isolates gene sequencing, since 16S rRNA gene sequence variation are not sufficient to differentiate Acinetobacter Direct culture of animal samples on agar plates yielded species.19 Amplification and analysis of the rpoB gene 12 isolates, which were presumptively identified as sequences were performed using a procedure described belonging to the genus Acinetobacter after phenotypic previously.4 An rpoB sequence similarity of 95% to a analysis of colony morphology, Gram staining, spore reference strain was considered as sufficient for identi- forming ability, respiratory type, motility, oxidase, fication at species level.4 catalase and API 20 NE tests (Table 1). Overall, Acinetobacter spp. was found in approximately 0.5% Antimicrobial susceptibility testing of the mice examined per year. They were not detectable in all microbiological units of the facility by using Antimicrobial susceptibility testing of the 12 routine health monitoring procedures described above. Acinetobacter isolates was performed by broth micro- Nevertheless, the isolates described here were found in dilution according to the human-specific Clinical & both breeding and experimental barriers. The strains, Laboratory Standards Institute (CLSI) document isolated mostly from cecum or the oropharynx, were M100-S.20 The decision to use the human-specific docu- subjected to 16S rDNA and rpoB gene sequencing. ment for testing the Acinetobacter isolates from mice Nine of the isolates possessed a 16S rDNA sequence was taken because there is no testing method for similarity 99% to that of a bacterial type strain Acinetobacter spp. available in the veterinary-specific deposited in GenBank, whereas the remaining three documents VET06 21 and VET08 .22 Moreover, neither strains showed a sequence similarity 97% but < 99% mouse-specific nor veterinary-specific clinical break- to that of a prototype strain (Table 1). The similarity points for Acinetobacter spp. are available.21,22 level of 16S rRNA genes allowed the classification of all Therefore, the human-specific clinical break- isolates into the genus Acinetobacter. The further rpoB points—when available—were used for categorization gene analysis permitted identification at species level for of the minimal inhibitory concentration (MIC) values all isolates. Overall, seven isolates belonged to the spe- as susceptible, intermediate, or resistant.20 Custom- cies Acinetobacter radioresistens, three isolates were made microtiter plates (MCS Diagnostics, Swalmen, Acinetobacter genomosp. 14BJ. The remaining two iso- The Netherlands) were used to test the Acinetobacter lates were classified as Acinetobacter pitii and isolates for their susceptibility to 23 antimicrobial Acinetobacter sp. ANC 4051, respectively (Table 1). agents/combinations (ampicillin, amoxicillin-clavulanic acid, ceftiofur, cefquinome, cephalothin, cefotaxime, Antimicrobial susceptibility of the cefoperazone, imipenem, tetracycline, doxycycline, gen- Acinetobacter isolates tamicin, neomycin, streptomycin, nalixic acid, ciprofloxacin, enrofloxacin, marbofloxacin, florfenicol, The MIC values of the 23 antimicrobial agents for the tilmicosin, tulathromycin tiamulin, colistin, Acinetobacter isolates tested are presented in Table 2. eg tal. et Benga

Table 2. Antimicrobial susceptibility of mice Acinetobacter species isolates.

MICs of antimicrobial agent (mg/L)

Isolate Species FFN IMI TIA COL NAL STR DOX NEO CIP GEN ENR SXT MAR TET TIL TUL AMP AUG2 XNL CEQ CEP FOT FOP

1300/07 A. radioresistens 16 0.25 8 0.25 8 2 0.12 0.5 0.12 0.5 0.06 0.5/9.5 0.12 1 16 1 8 4/2 4 0.5 64 4 16 1195/08 A. sp. ANC 4051 128 0.12 128 0.5 8 2 0.25 0.5 0.25 0.5 0.12 0.25/4.75 0.12 4 64 64 32 16/8 16 4 256 16 64 55/11 A. radioresistens 8 0.12 8 0.12 4 0.5 0.25 0.25 0.12 0.12 0.06 0.03/0.59 0.12 2 8 1 2 2/1 2 0.5 32 2 16 72/11 A. radioresistens 64 0.25 32 0.12 8 2 0.5 0.5 0.25 0.25 0.12 0.5/9.5 0.25 8 64 4 8 4/2 4 2 128 8 32 272/11 A. pitii 128 0.12 128 0.5 4 16 0.06 0.5 0.12 0.5 0.03 0.06/1.19 0.12 2 128 64 16 8/4 16 2 256 8 32 764/11 A. radioresistens 16 0.25 8 0.25 4 4 0.06 1 0.06 0.25 0.03 0.12/2.38 0.06 0.5 16 2 4 4/2 2 0.5 32 4 32 658/14 A. genomosp. 14BJ 64 0.5 128 1 2 16 0.25 1 0.12 1 0.06 0.5/9.5 0.12 4 128 64 16 16/8 8 2 256 4 32 1234/14 A. genomosp. 14BJ 32 0.25 128 1 2 8 0.25 1 0.06 1 0.06 0.5/9.5 0.03 4 128 64 16 8/4 8 1 128 4 32 1258/14a A. radioresistens 16 0.25 8 0.25 2 128 0.12 0.25 0.06 0.25 0.06 0.03/0.59 0.06 1 16 2 4 2/1 2 0.5 32 2 16 1258/14b A. genomosp. 14BJ 32 0.25 128 1 2 16 0.25 1 0.12 1 0.03 0.25/4.75 0.03 2 128 64 32 8/4 8 1 128 8 32 1301/14 A. radioresistens 16 0.25 8 0.12 4 256 0.06 0.5 0.12 0.25 0.06 0.03/0.59 0.12 1 16 2 4 4/2 2 0.5 16 4 32 1302/14 A. radioresistens 16 0.25 8 0.25 2 128 0.12 0.5 0.12 0.25 0.06 0.03/0.59 0.06 1 16 2 4 4/2 2 0.5 16 2 32

FFN: Florfenicol; IMI: imipenem; TIA: tiamulin; COL: colistin; NAL: nalidixic acid; STR: streptomycin; DOX: doxycycline; NEO: neomycin; CIP: ciprofloxacin; GEN: gentamicin; ENR: enrofloxacin; SXT: trimethoprim/sulfamethoxazole; MAR: marbofloxacin; TET: tetracycline; TIL: tilmicosin; TUL: tulathromycin; AMP: ampicillin; AUG2: amoxicillin/clavulanic acid; XNL: ceftiofur; CEQ: cefquinome; CEP: cephalothin; FOT: cefotaxime; FOP: cefoperazone. Susceptible and intermediate phenotypes according to CLSI clinical breakpoints (CLSI, 2018a) are indicated by a green and a yellow background, respectively. 473 474 Laboratory Animals 53(5)

The MIC distribution varied from three to five dilution laboratory mice from our institution. The prevalence steps for 21 of the 23 antimicrobial agents tested. of about 0.5% Acinetobacter spp. assumed in our However, a broad MIC distribution over seven and study might be underestimated, since the methods ten dilutions was recorded for tulathromycin and used here did not specifically target the isolation of streptomycin, respectively (Table 2). Relatively low Acinetobacter spp., but rather all FELASA-listed bac- MIC values (0.03–4.0 mg/L) were seen for the majority teria. The presence of Acinetobacter spp. in laboratory of the antimicrobial agents tested, whereas high MIC rodents is known among people performing microbio- values (up to 256 mg/L) were recorded for some other logical monitoring in rodent facilities. Despite this fact, antimicrobial agents. The lowest overall MIC values to date there is only one study reporting the isolation of were noted for enrofloxacin (0.03–0.12 mg/L), whereas Acinetobacter strains from laboratory mice and rats.11 high values (64 mg/L) were seen for, e.g., cephalothin, In that study, all strains isolated were classified as cefoperazone, tiamulin and tulathromycin. A. baumannii by phenotypical methods.11 However, For eight of the tested antimicrobial agents, namely phenotypical species identification among cefotaxime, imipenem, colistin, tetracycline, doxycyc- Acinetobacter isolates remains challenging.24 The geno- line, ciprofloxacin, gentamicin, and trimethoprim-sulfa- typic identification procedure used in our study allowed methoxazole, the CLSI-approved human-specific an exact identification to species level. Most of the spe- clinical breakpoints were applied.20 However, it cies found in our study belonged to A. radioresistens or should be noted that these breakpoints might not be Acinetobacter genomosp. 14BJ, but also isolates of appropriate to predict the clinical outcome for the A. pitii or Acinetobacter sp. ANC 4051, belonging to treatment of laboratory mice, due to possible differ- the A. calcoaceticus-baumannii complex, which consti- ences in the pharmacokinetic and pharmacodynamic tutes the most common causal agents of Acinetobacter parameters between humans and mice. Nevertheless, infections in hospitals worldwide, were documented they allow a prediction in case of an infection of (Table 1). The finding of these bacterial species points humans due to transmission of these Acinetobacter towards a potential risk of transfer of these bacteria strains from the laboratory mice. Using these human between animals and humans. The Acinteobacter clinical breakpoints, all isolates were susceptible to imi- strains were isolated from several parts of the bodies penem, colistin, doxycycline, gentamicin, trimetho- of the animals, including skin, upper respiratory tract, prim/sulfamethoxazole and ciprofloxacin.20 The low cecum and feces. We have recently shown that survival MIC values (0.03–0.25 mg/L) seen for enrofloxacin of A. radioresistens on animal bedding in a mouse room and marbofloxacin are in accordance with the suscep- easily exceeded 6 weeks,6 suggesting that this group of tibility to ciprofloxacin, since resistance to fluoroquino- bacteria is capable of persisting over long periods of lones is commonly mediated via the same mechanism.23 time in the mouse room environment, representing a The MICs for nalidixic acid ranged between 2 mg/L risk of infection for humans and animals. This finding and 8 mg/L. is in accordance with the findings that Acinetobacter In comparison to doxycycline, to which all isolates can survive several weeks on abiotic surfaces under were classified as susceptible, one isolate, 72/11, was dry conditions due to biofilm formation and the devel- classified as intermediate to tetracycline.20 Only strain opment of dormant cells.24 Nevertheless, all materials 1195/08 showed intermediate susceptibility to cefotax- used in the routine husbandry procedures in the mouse ime, while the remaining isolates were classified as sus- room, such as cage, bedding, feed, water, are sterilized ceptible.20 However, the MIC values for other before use, suggesting that the Acinetobacter isolates b-lactams such as cephalothin (8– 256 mg/L) and found were more probably transferred from the cefoperazone (16– 64 mg/L) were comparatively mouse into the environment rather than being picked high. The MICs for neomycin ranged from 0.25 mg/L up from environmental sources. to 1 mg/L and were relatively low. However, the MIC A further valuable finding is the antimicrobial sus- values for other substances, such as florfenicol ceptibility data of the Acinetobacter isolates (Table 2). (8–128 mg/L), tiamulin (8– 128 mg/L), and tilmicosin By using the human-specific clinical breakpoints for (8–128 mg/L) were comparatively high for most of the Acinetobacter spp., none of the isolates was classified isolates tested, suggesting that a therapy using these as resistant to imipenem, colistin, doxycycline, cipro- antimicrobial agents might be unsuccessful. floxacin, gentamicin, trimethoprim/sulfamethoxazole, tetracycline and cefotaxime.20 Moreover, only single Discussion isolates were classified as intermediate to tetracycline and cefotaxime, respectively. For 15 out of 23 (70%) In the present investigation, we analyzed the species antimicrobial agents used, no CLSI-approved clinical diversity of Acinetobacter spp. isolates obtained breakpoints were available. For antimicrobial agents during the routine health monitoring program of such as neomycin, enrofloxacin, and marbofloxacin, Benga et al. 475 the MIC values recorded were comparatively low promising, since resistant Acinetobacter isolates are a (1 mg/L), whereas for other substances such as florfe- global problem in human medicine.24 Especially resist- nicol, tiamulin, tilmicosin, cephalothin, and cefopera- ance to last resort antimicrobial agents such as carba- zone, they were comparatively high (up to 64 mg/L penems is a major threat for the treatment of to 256 mg/L) (Table 2). Such high antimicrobial con- nosocomial infections.24,25. centrations would be hard to achieved in vivo and, thus, In conclusion, we document that the laboratory render a therapeutic approach using these substances mouse can be involved in the extra-hospital epidemi- most likely unsuccessful. It should be noted that ology of Acinetobacter infections. Moreover, the colon- Acinetobacter are intrinsically resistant to certain anti- ization or infection of laboratory rodents with this microbial agents, and the CLSI document M10020 group of bacteria can be problematic, especially for stated that Acinetobacter baumannii and the immunocompromized laboratory animal breeds. Acinetobacter calcoaceticus complex are intrinsically resistant to penicillin, ampicillin, amoxicillin, amoxicil- Acknowledgments lin-clavulanic acid, first generation cephalosporins (cephalothin, cefazolin), second generation cephalo- We gratefully acknowledge Vivian Hensel, Isabel Scha¨fer, sporins (cefuroxime), cephamycins (cefoxitin, cefo- and Manuela Stockhausen for excellent technical assistance. tetan), clindamycin, daptomycin, fusidic acid, glycopeptides (vancomycin, teicoplanin), linezolid, Declaration of Conflicting Interests macrolides (erythromycin, azithromycin, clarithromy- The author(s) declared no potential conflicts of interest with cin), quinupristin-dalfopristin, rifampicin, ertapenem, respect to the research, authorship, and/or publication of this trimethoprim, chloramphenicol and fosfomycin. Due article. to the known intrinsic resistance of Gram-negative bacteria to some of these antimicrobial agents, some of Funding them, such as erythromycin, clindamycin, vancomycin, linezolid and quinupristin-dalfopristin, were not The author(s) received no financial support for the research, included in the test panel used. The comparably high authorship, and/or publication of this article. MICs observed for ampicillin and amoxicillin-clavulanic acid, as well as the high cephalothin MICs can be ORCID iD 20 explained by intrinsic resistance. In addition, the find- Laurentiu Benga http://orcid.org/0000-0002-4729-5964 ing of high florfenicol MICs, pointing towards a possible resistance, are in accordance with the intrinsic resistance reported for the related substance chloram- References phenicol.20 Multidrug-resistant Acinetobacter isolates 1. Munoz-Price LS and Weinstein RA. Acinetobacter infec- are common among hospital isolates.4 Resistance to tion. N Engl J Med 2008; 358: 1271–1281. antimicrobial agents of Acinetobacter animal isolates 2. Maragakis LL and Perl TM. Acinetobacter baumannii: is widespread, covering a broad resistance spectrum, epidemiology, antimicrobial resistance, and treatment options. Clin Infect Dis 2008; 46: 1254–1263. including gentamicin, tetracycline, and enrofloxacin, 10 3. Rafei R, Hamze M, Pailhories H, et al. Extrahuman epi- among others. Laboratory animals are not usually demiology of Acinetobacter baumannii in Lebanon. Appl exposed to antibiotic treatments, which would trigger Environ Microbiol 2015; 81: 2359–2367. the selection of resistant strains. Nevertheless, therapy 4. Choi JY, Kim Y, Ko EA, et al. 2012. Acinetobacter spe- with antimicrobial agents might be indicated in order to cies isolates from a range of environments: species survey save valuable unique mice strains until rederivation by and observations of antimicrobial resistance. Diagn other methods is possible. Goo and co-workers tested Microbiol Infect Dis 2012; 74: 177–180. the resistance of several rodent Acinetobacter isolates to 5. Houang ET, Chu YW, Leung CM, et al. Epidemiology a restricted group of antimicrobial agents and found and infection control implications of Acinetobacter spp. them resistant to cefazolin, cefoxitin and nitrofuran- in Hong Kong. J Clin Microbiol 2001; 39: 228–234. toin, but susceptible to other commonly used anti- 6. Benga L, Benten WP, Engelhardt E, et al. Survival of biotics.11 However, the use of the human-specific bacteria of laboratory animal origin on cage bedding and inactivation by hydrogen peroxide vapour. Lab clinical breakpoints also allows a prediction of a thera- Anim 2017; 51: 412–421. peutic success in the case of human infection with such 7. Espinal P, Marti S and Vila J. Effect of biofilm formation a mouse-associated Acinetobacter isolate, which might on the survival of Acinetobacter baumannii on dry sur- occur after transmission to an immunocompromized faces. J Hosp Infect 2012; 80: 56–60. person. The finding that these strains were comparably 8. Poirel L, Bercot B, Millemann Y, et al. Carbapenemase- susceptible to most of the antimicrobial agents for producing Acinetobacter spp. in Cattle, France. Emerg which clinical breakpoints are available seems Infect Dis 2012; 18: 523–525. 476 Laboratory Animals 53(5)

9. Smet A, Boyen F, Pasmans F, et al. OXA-23-producing environmental and clinical unidentifiable bacterial iso- Acinetobacter species from horses: a public health lates. J Clin Microbiol, 200; 38: 3623–3630). hazard? J Antimicrob Chemother 2012; 67; 3009–3010. 19. La Scola B, Gundi VA, Khamis A, et al. Sequencing of 10. Zordan S, Prenger-Berninghoff E, Weiss R, et al. the rpoB gene and flanking spacers for molecular identi- Multidrug-resistant Acinetobacter baumannii in veterin- fication of Acinetobacter species. J Clin Microbiol 2006; ary clinics, Germany. Emerg Infect Dis 2011; 17: 44: 827–832. 1751–1754. 20. Clinical and Laboratory Standards Institute. 11. Goo JS, Jang MK, Shim SB, et al. Monitoring of anti- Performance standards for antimicrobial susceptibility. biotic resistance in bacteria isolated from laboratory ani- CLSI document M100-S. 28th ed. Wayne, PA: Clinical mals. Lab Anim Res 2012; 28: 141–145. and Laboratory Standards Institute, 2018a. 12. Mu¨ller S, Janssen T and Wieler LH. Multidrug resistant 21. Clinical and Laboratory Standards Institute. Methods for Acinetobacter baumannii in veterinary medicine emer- antimicrobial susceptibility testing of infrequently isolated gence of an underestimated pathogen? Berl Munch or fastidious bacteria isolated from animals. CLSI docu- Tierarztl Wochenschr 2014; 127: 435–446. ment VET06, 1st ed. Wayne, PA: Clinical and 13. Ma¨hler M, Berard M, Feinstein R, et al. FELASA rec- Laboratory Standards Institute, 2017. ommendations for the health monitoring of mouse, rat, 22. Clinical and Laboratory Standards Institute. hamster, guinea pig and rabbit colonies in breeding and Performance standards for antimicrobial disk and dilution experimental units. Lab Anim 2014; 48: 178–192. susceptibility tests for bacteria isolated from animals. CLSI 14. Nicklas W, Baneux P, Boot R, et al. Recommendations for document VET08, 4th ed. Wayne, PA: Clinical and the health monitoring of rodent and rabbit colonies in Laboratory Standards Institute, 2018b. breeding and experimental units. Lab Anim 2002; 36: 20–42. 23. Clinical and Laboratory Standards Institute. Generation, 15. Benga L, Benten WP, Engelhardt E, et al. Analysis of presentation, and application of antimicrobial susceptibility 16S-23S rRNA internal transcribed spacer regions in test data for bacteria of animal origin; a report. CLSI Pasteurellaceae isolated from laboratory rodents. document VET05RE (formerly X08-R). Wayne, PA: J Microbiol Methods 2012; 90: 342–349. Clinical and Laboratory Standards Institute, 2011. 16. Benga L, Benten WP, Engelhardt E, et al. 16S ribosomal 24. Kim UJ, Kim HK, An JH, et al. Update on the epidemi- DNA sequence-based identification of bacteria in labora- ology, treatment, and outcomes of carbapenem-resistant tory rodents: a practical approach in laboratory animal Acinetobacter infections. Chonnam Med J 2014; 50: bacteriology diagnostics. Lab Anim 2014; 48: 305–312. 37–44. 17. Cai H, Archambault M and Prescott JF. 16S ribosomal 25. Garnacho-Montero J, Amaya-Villar R, Ferra´ndiz-Millo´n RNA sequence-based identification of veterinary clinical C, et al. Optimum treatment strategies for carbapenem- bacteria. J Vet Diagn Invest 2003; 15: 465–469. resistant Acinetobacter baumannii bacteremia. Expert Rev 18. Drancourt M, Bollet C, Carlioz A, et al. 16S ribosomal Anti Infect Ther 2015; 13: 769–777. DNA sequence analysis of a large collection of

Re´sume´ L’e´pide´miologie des infections extra-hospitalie`res a` Acinetobacter fait l’objet de de´bats. Au cours des dernie`res anneés, la pre´valence des infections animales a` Acinetobacter multire´sistantes a augmente´ conside´rablement. L’objectif de la pre´sente e´tude e´tait de prećiser les espe`ces d’Acinetobacter isoleés de souris de laboratoire et de tester leur sensibilite´ aux antimicrobiens. Au cours d’un suivi microbiologique routinier de souris de laboratoire, douze espe`ces d’Acinetobacter ont e´te´ isoleés. Le se´quençage du ge`ne codant rpoB et de l’ARNr 16S a permis l’identification de sept des isolats comme Acinetobacter radioresistens, trois isolats appartenaient a` l’espe`ce geńomique Acinetobacter 14BJ, un isolat e´tait classe´ comme Acinetobacter pitii et un comme appa- rtenant a` l’espe`ce AcinetobacterANC 4051. Les valeurs de distribution de la concentration minimale inhibitrice (CMI) e´taient uniformes pour 21 des 23 agents antimicrobiens teste´s, tandis qu’une distribution large de la CMI e´tait enregistreé pour la tulathromycine et la streptomycine. Les valeurs de CMI enregistreés e´taient faibles pour la majorite´ des antibiotiques teste´s. Neánmoins, des valeurs de CMI tre`se´leveés ont e´te´ enregistreés pour le florfeńicol, la tiamuline, la tilmicosine et la ce´phalothine dans la plupart des isolats, ce qui rendra probable- ment infructueuse toute approche the´rapeutique utilisant ces substances. En conclusion, nous documentons la colonisation de la souris de laboratoire avec diffe´rentes espe`ces d’Acinetobacter, montrant des profils de sensibilite´ aux antibiotiques similaires, avec des implications possibles au niveau de l’e´pide´miologie d’Acinetobacter ainsi que de l’e´levage et de l’expe´rimentation des animaux colonise´s.

Abstract Die außerklinische Epidemiologie von Acinetobacter-Infektionen ist ein Gegenstand der Debatte. In den letz- ten Jahren ist die Pra¨valenz von tierischen multiresistenten Acinetobacter-Infektionen deutlich gestiegen. Benga et al. 477

Ziel der vorliegenden Studie war es, aus Labormaüsen isolierte Acinetobacter-Arten zu bestimmen und sie auf ihre Empfindlichkeit gegenu¨ber antibakteriellen Wirkstoffen zu testen. Bei der routinema¨ßigen mikrobio- logischen U¨berwachung von Labormaüsen wurden zwo¨lf Acinetobacter spp. isoliert. Mittels 16S rRNA und rpoB Gensequenzierung wurden sieben der Isolate als Acinetobacter radioresistens identifiziert, drei Isolate geho¨rten zur Acinetobacter Genomospezies 14BJ, ein Isolat wurde als Acinetobacter pittii und eines als Acinetobacter sp. ANC 4051 klassifiziert. Die Verteilung der Werte der minimalen Hemmkonzentration (MIC) war bei 21 der 23 getesteten antibakterielle Wirkstoffe einheitlich, wa¨hrend fu¨r Tulathromycin und Streptomycin eine breite MIC-Verteilung festgestellt wurde. Die MIC-Werte waren fu¨r die Mehrheit der getesteten Antibiotika niedrig. Allerdings wurden fu¨r Florfenicol, Tiamulin, Tilmicosin und Cephalothin bei den meisten Isolaten sehr hohe MIC-Werte verzeichnet, weshalb ein Therapieansatz mit diesen Substanzen wahrscheinlich erfolglos sein wird. Mit dieser Arbeit dokumentieren wir die Besiedelung von Labormaüsen mit verschiedenen Acinetobacter- Arten, die a¨hnliche Antibiotika-Empfindlichkeitsprofile aufweisen, sowie mo¨gliche Auswirkungen auf die Acinetobacter-Epidemiologie und auf die Haltung der besiedelten Tiere und Versuche mit ihnen.

Abstract La epidemiologıá extrahospitalaria de las infecciones por Acinetobacter es un tema de debate. En los u´ltimos anõs, la prevalencia de infecciones por Acinetobacter resistentes a mu´ltiples fa´rmacos en animales aumento´ considerablemente. El objetivo del presente estudio fue especificar las especies de Acinetobacter aisladas de ratones de laboratorio y evaluarlas para determinar su susceptibilidad antimicrobiana. Durante la comproba- cioń microbiolo´gica de rutina de ratones de laboratorio, doce Acinetobacter spp. fueron aislados por medio de la secuenciacioń de los genes 16S rRNA y rpoB, siete de los aislamientos se identificaron como Acinetobacter radioresistens, tres aislamientos pertenecieron a Acinetobacter genoespecie 14BJ, un aislado se clasifico´ como Acinetobacter pitii y uno como Acinetobacter sp. ANC 4051. La distribucioń de los valores de concen- tracioń inhibitoria mıńima (CIM) fue uniforme para 21 de los 23 agentes antimicrobianos analizados, mientras que se registro´ una amplia distribucioń de CIM para tulatromicina y estreptomicina. Los valores de CIM registrados fueron bajos para la mayorıá de los antibio´ticos probados. Sin embargo, en la mayorıá de los aislamientos, se registraron valores muy altos de CIM, que probablemente harań que un enfoque terapeútico con el uso de estas sustancias no tenga e´xito, para florfenicol, tiamulina, tilmicosina y cefalotina. En con- clusioń, documentamos la colonizacioń de ratones de laboratorio con diferentes especies de Acinetobacter, mostrando perfiles de susceptibilidad a antibio´ticos similares, con posibles implicaciones en la epidemiologıá de Acinetobacter, ası´ como en la crıá y experimentacioń de los animales colonizados. Original Article Laboratory Animals 2019, Vol. 53(5) 478–490 ! The Author(s) 2018 Reliability of behavioral tests in the middle Article reuse guidelines: sagepub.com/journals- cerebral artery occlusion model of rat permissions DOI: 10.1177/0023677218815210 journals.sagepub.com/home/lan Junghoon Yu1 , Jinkyoo Moon1, Joonyoung Jang1, Jee In Choi2, Jooeun Jung3, Sunyoung Hwang2 and MinYoung Kim1,2

Abstract Stroke is one of the leading causes of death and disability worldwide, and its incidence is increasing. To overcome impairment from stroke, translational research for developing new therapeutic technologies has been conducted and middle cerebral artery occlusion (MCAo) in rat is the representative model. Since recovery from neurological impairment in contralateral limbs caused by brain damage is the major goal of treatment, behavioral tests that assess the relevant function are used. To determine therapeutic effect, obtaining reliable results of behavioral assessment is a prerequisite. However, studies on the reliability of behavioral tests in the MCAo rat model and necessity of prior training have not yet been reported. In this study, the authors investigate relative and absolute inter-rater reliabilities of modified neurological severity score (mNSS), cylinder test, and grid-walking test before training and repeated training every week until the reliability of results reached a satisfactory level. The training included repeated learning of the scoring system and decreasing disagreements among the raters. For MCAo modeling, adult male Sprague-Dawley rats were subjected to 90 min of transient MCAo. Six raters conducted behavioral tests via observation of video-recording on sham-operated and MCAo model rats at 3 or 7 days after the intervention. An independent experimenter randomly numbered each video clip to blind the experiment. The results of reliabilities were unacceptable before training and improved to a satisfactory level after 6 weeks of training in all of the tests. In conclusion, mNSS, cylinder test, and grid-walking test on the MCAo rat model are reliable evaluation methods after conducting appropriate training.

Keywords MCAo, behavioral test, reliability, rodents, stroke

Date received: 26 June 2018; accepted: 24 October 2018

Introduction cerebrovascular physiology and anatomy of rat is similar to that of humans.6 Completed stroke means continued neurologically impaired status from cerebrovascular events including ischemia and hemorrhage, and it is the second leading 1Department of Rehabilitation Medicine, CHA University School of cause of death.1,2 As damage to brain tissue remains Medicine, Seongnam, Republic of Korea 2Rehabilitation and Regeneration Research Center, CHA diﬃcult to repair, much therapeutic eﬀort has been University, Seongnam, Republic of Korea made with fundamentals from translational research 3Department of Neurology, University of Texas Health Science 3 using animal models. Center at Houston, Houston, Texas, USA To date, the experimental focal transient ischemic stroke model by middle cerebral artery occlusion Corresponding author: MinYoung Kim, MD, PhD, Department of Rehabilitation Medicine, (MCAo) in rat is the model most commonly used for 4,5 CHA Bundang Medical Center, CHA University School of Medicine, mimicking human stroke. Using rat has advantages Seongnam 13496, Korea. for cost and ethical reasons. Moreover, the Email: [email protected] Yu et al. 479

One of the major sequelae of stroke in humans is the Materials and methods 7,8 paralysis of contralateral extremities. The MCAo rat Animals model also reveals paralytic features, and thus an important target for therapy is usually motor and/or All experimental protocols were approved by the sensory recovery in use of the limbs.9 There are several Institutional Animal Care and Use Committee of the behavioral tests used in the MCAo rat model to evalu- study institute. Adult male Sprague-Dawley rats were ate neurological impairment in the contralateral limbs, purchased from Charles River Laboratories (Seoul, such as the modified neurological severity score Korea). The rats were housed in a temperature-con- (mNSS), cylinder test, grid-walking test, accelerated trolled room (22C 2), with constant humidity rotarod, and grip strength test.9–15 (50% 10%), and a constant supply with purified As MCAo rat model analysis is the most important water and food ad libitum on 12-hour night–day reference for approving the effect of a specific therapy, cycle. The Sprague-Dawley rats usually weighed responses in behavioral tests have great significance.16 250–300 g (8 weeks old) at the time of surgery. The Therefore, acquiring reliability of behavioral tests is rats were adapted to the environment for at least 1–2 necessary.17 Although reliability evaluation is widely days before surgery. Rats were fed feed and water freely used for brain-injured human subjects, including in until the evening before surgery. Feeds were stopped stroke,18-20 it has not yet been reported for the behavior from the night before surgery until the day of surgery, tests in the MCAo rat model.21,22 but water was continuously supplied. ‘‘Reliability’’ means consistency and stability of scores on an assessment tool, and it is classified into 23 Modeling transient middle cerebral artery relative and absolute reliabilities. Inter-rater reliabil- occlusion ity is an estimate of how consistently different raters use the test on the same subject.19 Although ‘‘relative reli- Transient MCAo was induced by an intraluminal vas- ability’’ is the most commonly used tool in reliability cular occlusion modified in our laboratory.30 Rats studies, measurement errors between repeated measure- were initially anesthetized with 3.0% isoflurane in ments by the other rater can be quantified with ‘‘abso- 70% N2O and 30% O2 (v/v). Under an operating lute reliability.’’24,25 Relative reliability expresses microscope, a midline neck incision was made with agreement in order of scores in a group and not the scissors between the manubrium and the jaw. The absolute value of the score,26 and the intraclass correl- right common carotid artery (CCA) was separated ation coefficient (ICC) is widely used to quantify the and isolated from the vagus nerve and temporarily relative reliability.26 Even when the ICC is high ligated during the whole period of occlusion. The enough, it does not ensure agreement in absolute right external carotid artery (ECA) and the right inter- values of scores.24 The absolute reliability is derived nal carotid artery (ICA) were also isolated. The right by calculating the difference among the scores from ECA was ligated and cauterized to make an ECA the raters; the most well-known indicators are standard stump. The hypothalamic artery was also ligated. A error measurement (SEM) and smallest real difference small hole was made with micro scissors in the ECA (SRD).20 The SEM represents standard deviation (SD) stump. A nylon filament suture with a silicon-coated of measurement errors and the SRD signifies sensitivity tip was inserted through the hole in the ECA. To to detect change in the scale.24,27 Therefore, securing steadily hold the filament and effectively occlude the successful values of ICC, SEM, and SRD is important MCA, the ICA was ligated temporarily. The ECA to guarantee reliable results of a test. stump over the hole was temporarily ligated with a Clinical studies usually offer a training program silk suture in order to stop bleeding from the hole. before conducting a test in order to secure reliabil- After 90 min of occlusion, the rats were re-anesthe- ity.18,19 However, the necessity of training programs tized and the suture was gently removed from the for behavioral tests in the MCAo rat model has not MCA to re-perfuse the MCA territory. The hole in yet been reported, although guidelines have been ECA stump was permanently ligated and the tempor- provided.28,29 ary ligation in the CCA was removed to allow regain- In this study, the authors investigated the relative ing of blood flow to the brain. The midline neck and absolute reliabilities of behavioral tests in the incision was closed and injection of 3 ml of intraper- MCAo rat model using mNSS, cylinder test, and itoneal saline was provided to prevent dehydration. As grid-walking test. The study also aimed to verify a control group, sham-operated rats were subjected to whether there is a necessity to train the raters, by con- the same procedure without MCAo. The rats used for ducting reliability evaluation prior to and during the behavioral tests were randomly selected from sham- training until the indices of the reliabilities were operated and MCAo rats at 3 days and 7 days after satisfactory. surgery. 480 Laboratory Animals 53(5) Behavioral tests Cylinder test Modified neurological severity score. On the mNSS, On the cylinder test, asymmetric use of forepaw was neurological functions including motor and sensory analyzed by video-recording of the rat in a transparent systems as well as reflexes and balance are graded on cylinder (20 cm in diameter and 30 cm in height).36 a numeric scale from 0 to 18.31 One experimenter per- A mirror was placed under the cylinder at a 45 angle formed the mNSS and another experimenter video- to video-record (Figure 2). The rat was placed by an recorded it to evaluate the reliability of multiple experimenter in the cylinder, and the vertical exploratory raters. The scoring method follows the description in behavior was observed. Movement of the rat getting up Table 1 by observation of abnormal asymmetric and placing its forepaws on the wall 20 times in total was responses in the limbs of the impaired side.31 The used for rating. For the analysis, the video was played at higher score indicates more severe neurological impair- 0.3 times slower than real time. During a rear, the first ment from the unilateral cerebral lesion.32 The original forepaw to place the wall was recorded as right and left. mNSS consists of sub-tests including motor tests When one forepaw was placed on the floor and other (raising rat by tail, walking on floor), sensory tests (pla- forepaw was on the wall, it was not counted. In cases of cing test, proprioceptive test), beam balance test, reflex both forepaws touching the wall at the same moment or absence and abnormal movements (Figure 1). a forepaw touching the wall followed by the other fore- However, in this study, reflex absence and abnormal paw without interval, the rater recorded ‘‘bilateral.’’ movements were excluded because they are irrelevant Exploring the wall after initial touching was not rec- to damage in the MCA area.10 Therefore, in this study, orded. In order to count the next contact, both forepaws zero points stands for normal and 14 was the score of should have touched the floor after the initial placing on the most severe neurological defect (Table 1).29,33-35 the wall. The number of contacts consisting of ipsilateral Although not mentioned in previous studies,31,33 (intact side), contralateral (impaired), or bilateral con- 1 point was given when there was no reaction more tacts were counted. An asymmetry score was made by than three times out of five attempts, in order to the calculation formula as ðipsilateral þ bilateral=2Þ= increase the accuracy in the sensory tests in this ðipsilateral þ contralateral þ bilateral Þ: research.

Table 1. Modified neurological severity score (mNSS).

Tests Points

Raising rat by tail (normal ¼ 0; maximum ¼ 3) Flexion of forelimb 1 Flexion of hindlimb 1 Head moved > 10 to vertical axis within 30 s 1 Walking rat on floor (normal ¼ 0; maximum ¼ 3) Normal walk 0 Inability to walk straight 1 Circling toward the paretic side 2 Falls down to paretic side 3 Beam balance test (normal ¼ 0; maximum ¼ 6) Balances with steady posture (>60 s) 0 Grasps side of the beam 1 Hugs beam and 1 limb falls down from beam 2 Hugs beam and 2 limbs falls down from beam, or spins on beam (>60 s) 3 Attempts to balance on beam but falls off (>40 s) 4 Attempts to balance on beam but falls off (>20 s) 5 Falls off, no attempt to balance or hang on the beam (<20 s) 6 Sensory tests (normal ¼ 0; maximum ¼ 2) Placing test (visual and tactile test, try 5 times and have not moved more than 3 times) 1 Proprioceptive test (deep sensation, pushing the paw against the table edge to stimulate 1 limb muscles, try 5 times and have not moved more than 3 times) Maximum Points 14 Yu et al. 481

Figure 1. Modified Neurological Severity Score (mNSS). (a) Raising rat by tail; (b) Beam balance test; (c) Placing test; and (d) Proprioceptive test.

Figure 2. Cylinder Test. Spontaneous forelimb use of rat being assessed.

The cylinder test is advantageous in that it is the top of the cylinder, and putting another rat in the objective, easy, and sensitive to chronic deﬁcits cylinder for a while.37 which cannot be detected by other methods.10 However, the Sprague-Dawley rats used in this study Grid-walking test show inactivity when placed in a cylinder. Therefore, in this study, we used ways to encourage movement In the grid-walking test, an elevated metal square grid in the rats that did not aﬀect the asymmetry (40 40 cm2, with each grid cell 3 3cm2; height: score. These methods include picking up and 41 cm) was used.38 A video camera for assessment of replacing the rat into the cylinder, placing food in forepaw slip was located under the grid-walking 482 Laboratory Animals 53(5)

Figure 3. Grid-walking test. Spontaneous motor deficits and limb movements of rat being assessed. apparatus with an angle between 20 and 40 the published protocols, consensus had to be reached for (Figure 3). The rat was placed on the center of the some parts of the rating processes, as described in the apparatus by an experimenter, and allowed to explore results section. After achieving a satisfactory level of for 5 min. The behaviors of the rat on the grid were reliability, the last assessment for reliability was con- recorded with video camera and the motions of both ducted on 20 sham-operated rats and 40 MCAo rats forepaws were analyzed by raters. For the analysis, the without grouping again for confirmation. For blinding video was played at 0.3 times slower than real time. The of the experiment, the videos of sham-operated rats and slip of forepaw was scored when (a) the forepaw MCAo rats were randomly numbered by an independent entirely missed a rung at the time of stepping on the provider who was not part of the rater team at the rung, (b) the forepaw fell down between the rungs, or 6 weeks’ training session and the last reliability assess- (c) the forepaw slipped off during weight bearing.38 The ment. After random numbering, the six raters assessed case in which a rat placed its forepaws on the rung the behavioral tests. Through the random numbering, without slip was considered as a step. The analysis the six raters did not know whether the video was a was performed until the summation of steps and slips sham-operated or MCAo rat during in the scoring of both forepaws was 100. A percentage of affected-side process. forepaw slip among the summation of steps and slips of both forepaws was calculated. Statistical analysis

Assessment of behavioral tests The ICC value with 95% confidence interval was used to evaluate the relative reliability of each test, and a Six raters participated in this study. The experience of value of at least 0.90 for ICC was targeted to acquire the raters ranged from 1 month to 5 years. After review- a satisfactory level of relative reliability.39 The SEM ing the literature on behavioral tests, the initial guide- and SRD values were calculated to analyze absolute lines were made by the most experienced reliability for each test. Thep calculation formula for rater.10,13,31,32,38 In the first week, video-recordings for SEM was: SEM ¼ SD ð1 rÞ.40 In this study, five sham-operated rats and 20 MCAo rats were assessed ICC values were used as r values.41 The smaller SEM without grouping. The results were analyzed to find out means that the reliability of the measurement is higher. disagreement among raters. After that, five new MCAo 41 In the case of SEM values being less than 10% of the rats were weekly analyzed individually, and weekly meet- total average score, it was suggested that such SEM 23 ings were held thereon from the second week to the sixth values are acceptable. The SRDp was calculated by week. The causes of error and the disagreements among the formula nbs SRD ¼ 1:96 2 SEM.42 The the raters were identified. Although the raters followed target value of acceptable SRD was less than 10% of Yu et al. 483 the highest score.26 All statistical analyses were per- relative reliability, ICCs, for total mNSS, cylinder formed using SPSS version 21.0 software (SPSS Inc, test, and grid-walking test reached the target level Chicago, Illinois). from the fourth week of training (ICCs > 0.9, p < 0.001), the indicators of absolute reliability, SEM, and SRD reached the target from the third to fifth week Results of training (Figure 4). Difficulties that required consensus during Regarding the results of sub-tests of mNSS, ICCs the training became greater than 0.9 in ‘‘raising the rat by the tail,’’ ‘‘sensory tests,’’ and ‘‘beam balance test’’ from Published criteria for mNSS were sufficient, therefore the fourth week of training, and in ‘‘walking on the no further discussion was needed.31,33 However, in the floor’’ from the sixth week of training (p < 0.001). In case of the cylinder test, disagreement among the raters most sub-tests, SEM reached the target from the fifth occurred. The following conditions were observed: (1) a week of training and SRD reached the target from the part of the rat’s forepaw was touched on the wall and fourth week of training. Only ‘‘walking on the floor’’ (2) rats stood up at an angle not perpendicular to the reached the target at the sixth week of training for ground. SEM, while it took 5 weeks for SRD (Figure 5). After discussion, a score was given only when (1) the forepaw completely touched the wall, and (2) the rat Reliability of mNSS test after 6 weeks’ stood up perpendicular to the ground. A similar debate training occurred in the grid-walking test. Unexpected behaviors of rats were observed, such as (1) climbing up The ICC, SEM, and SRD values of the mNSS in 20 and down the wall around the grid apparatus and (2) sham-operated rats and 40 MCAo rats after training grabbing the edge of the grid. These were solved by are presented in Table 3. ICC showed excellent relative step-counting in the case of climbing down from the reliability in the sham-operated rats and the MCAo rats wall, and not as step-counting in climbing up the wall for total and all sub-tests (ICC 0.9, p < 0.001). As for and grabbing the outer edge. absolute reliability, SEMs were less than 10% of means, and SRDs were less than 10% of highest Reliability values at initial training session scores in the sham-operated rats and the MCAo rats for total and all sub-tests of the mNSS. The ICC, SEM, and SRD values of the mNSS, cylinder test, and grid-walking test in 20 MCAo rats and five Reliability of cylinder test and grid-walking sham-operated rats at the initial training session are pre- test after 6 weeks’ training sented in Table 2. The ICC for all measures showed unacceptable relative reliability (ICCs < 0.8, p < 0.001). The ICC, SEM, and SRD values of the cylinder test As for absolute reliability, the SEM values were greater and the grid-walking test in 20 sham-operated rats and than 10% of the mean value of mNSS and cylinder test. 40 MCAo rats after training are shown in Table 4. The The SRD were also greater than 10% of highest score of values of ICC showed excellent relative reliability in the mNSS, cylinder test, and grid-walking test. sham-operated rats and the MCAo rats for the cylinder test and grid-walking test (ICC 0.9, p < 0.001). Changes of reliability values during training Regarding absolute reliability, the values of SEM were less than 10% of means, and the values of SRD According to the plan and weekly results, the training were less than 10% of highest scores in the sham-oper- continued to the sixth week. While the indicator of ated rats and the MCAo rats.

Table 2. Reliability for mNSS, cylinder test, and grid-walking test in ischemic stroke model and sham-operated rats at initial training session (six raters).

Mean SD Range of scores (ICC:95% CI) p SEM SRD mNSS 7.408 2.961 0–14 0.775 (0.577–0.899) <0.001 1.404 3.893 Cylinder test 30.694 21.253 0–85 0.789 (0.604–0.905) <0.001 9.763 27.061 Grid-walking test 12.982 1.958 10–23 0.749 (0.529–0.887) <0.001 0.981 2.719

SD: standard deviation; ICC (95% CI): Intraclass Correlation Coefficient (95% Confidence Interval); SEM: Standard Error Measurement; SRD: Smallest Real Difference; mNSS: modified neurological severity score. Number of ischemic stroke rats used n ¼ 20, and sham- operated rats used n ¼ 5. 484 Laboratory Animals 53(5)

Figure 4. Changes in reliabilities of modified neurological severity score (mNSS), cylinder test, and grid-walking test during training. (a) ICC value, (b) SEM/Average, and (c) SRD/Measured highest score value. ICC (95% CI), Intraclass Correlation Coefficient (95% Confidence Interval); SEM, Standard Error Measurement; SRD, Smallest Real Difference. n ¼ 5 MCAo rats were used each time. Yu et al. 485

Figure 5. Changes in reliabilities of sub-tests of modified Neurological Severity Score during training. (a) ICC value, (b) SEM/Average, and (c) SRD/Measured highest score value. ICC (95% CI), Intraclass Correlation Coefficient (95% Confidence Interval); SEM, Standard Error Measurement; SRD, Smallest Real Difference. n ¼ 5 MCAo rats were used each time. 486 Laboratory Animals 53(5)

Table 3. Reliability for mNSS in sham-operated and MCAo rats after 6 weeks’ training (six raters).

Range of Mean SD scores ICC(95% CI) p SEM SRD

Raising rat by tail test Sham-operated rats 0.358 0.158 0 - 2 0.951 (0.908–0.978) <0.001 0.035 0.097 MCAo rat 1.454 0.238 0 - 3 0.933 (0.895–0.961) <0.001 0.061 0.170 Walking on floor test Sham-operated rats 0.042 0.014 0 - 1 0.960 (0.812–0.955) <0.001 0.003 0.008 MCAo rat 0.525 0.219 0 - 3 0.944 (0.912–0.967) <0.001 0.052 0.144 Beam balance test Sham-operated rats 0.433 0.189 0 - 3 0.961 (0.927–0.982) <0.001 0.037 0.103 MCAo rat 2.658 0.274 0 - 6 0.991 (0.987–0.995) <0.001 0.026 0.072 Sensory tests Sham-operated rats 0.342 0.014 0 - 2 0.996 (0.992–0.998) <0.001 0.001 0.002 MCAo rat 0.992 0.746 0 - 3 0.992 (0.987–0.995) <0.001 0.067 0.185 Total score Sham-operated rats 1.175 0.317 0 - 4 0.960 (0.925–0.982) <0.001 0.063 0.176 MCAo rat 5.629 0.590 0 - 12 0.991 (0.986–0.995) <0.001 0.056 0.155

Table 4. Reliability for cylinder test and grid-walking test in sham-operated and MCAo rats after 6 weeks’ training (six raters).

Range of Mean SD scores ICC(95% CI) p SEM SRD

Cylinder test Sham-operated rats 36.805 4.271 0–82.5 0.950 (0.950–0.978) <0.001 0.955 2.647 MCAo rats 28.673 2.584 0–65 0.994 (0.988–0.998) <0.001 0.200 0.555 Grid-walking test Sham-operated rats 4.808 4.533 0–16 0.989 (0.978–0.995) <0.001 0.475 1.318 MCAo rats 8.556 8.556 1–54 0.99 (0.989–0.997) <0.001 0.716 1.984

SD: standard deviation; ICC (95% CI): Intraclass Correlation Coefficient (95% Confidence Interval); SEM: Standard Error Measurement; SRD: Smallest Real Difference. Number of ischemic stroke rats used n ¼ 20, and sham-operated rats used n ¼ 40.

Discussion The behavioral tests have been used to evaluate efficacy of a possible therapy for stroke.31,45 Based on the In this study, relative and absolute inter-rater reliabil- evidence from animal experiments, including behav- ities of behavioral tests were evaluated for the MCAo ioral tests, clinical trials are underway.16,46 Thus, in a rat model, and changes in the reliabilities during the pre-clinical study using MCAo rat model, it would be training were also described. To our knowledge, this important to ensure reliability of the behavioral tests. is the first study that shows reliability evaluation of According to our results, pre-evaluation training for behavioral tests in rat with brain injury. As assessment raters in the behavioral test is necessary. The indices of tools, the mNSS, cylinder test, grid-walking test, accel- relative and absolute reliabilities of behavioral tests erated rotarod, and grip strength test were chosen after prior to the training were unacceptable. However, reli- reviewing previous studies. As preliminary results of the ability of the tests improved by conducting training. rotarod and grip strength test were poor, these were Ideally, a good scale does not need additional criteria excluded. The tests used in this study are the most rep- from the published version. Although the authors made resentative tools for evaluating neurological impair- efforts to follow the published protocol, we found diffi- ment status of the rat stroke model.9-15 The mNSS is culties in reaching agreement. In the cylinder test, area the one of the most frequently used comprehensive of forepaw touching and angle of trunk at rearing were scales for the MCAo rat model.43 The cylinder test the main problems of disagreement, and in the grid- evaluates unilateral motor impairment from stroke by walking test unexpected motion at the edge of the observing spontaneous forelimb use in rats,44 and the grid needed criteria for scoring. grid-walking test is a relatively simple and objective It took 4–6 weeks to obtain the target level of reli- tool to assess the accuracy of limb movement after ability in each test. In terms of relative reliability, we set stroke.10 the target value of ICCs as 0.90. Although ICC of at Yu et al. 487 least 0.70 was suggested for group comparisons,47 more Declaration of Conflicting Interests stringent criteria of ICC 0.90 was selected because The author(s) declared no potential conflicts of interest with this criterion is recommended for methods to make respect to the research, authorship, and/or publication of this 39 clinical decisions. In our study, the SEM and SRD article. were used additionally to evaluate the absolute reliability.20 To obtain the target level of absolute reliability, 3–5 weeks of training were required for total mNSS and Funding most of its sub-tests, cylinder test, and grid-waking test, The author(s) disclosed receipt of the following financial sup- and 6 weeks for a sub-test of the mNSS, ‘‘walking on port for the research, authorship, and/or publication of this the floor.’’ Final assessment, which measured 40 MCAo article: This work was supported by the Industrial Strategic Technology Development Program (10051152) funded by the model rats and 20 sham-operated rats, showed fulfill- Ministry of Trade, Industry & Energy (MOTIE, Korea). ment of reliability level for all behavioral tests. In clinical studies, training such as workshops and teaching of the evaluation tool are offered before evalu- ORCID iD ation. An evaluation tool for motor behavior in chil- Junghoon Yu http://orcid.org/0000-0001-6039-5364 dren with cerebral palsy was accompanied by a 30-hour MinYoung Kim http://orcid.org/0000-0001-5481-2985 workshop before confirming reliability.19 For evaluation of Parkinson’s disease, a teaching videotape of the Unified Parkinson’s Disease Rating Scale was References reviewed by researchers before inter-rater reliability 1. Organization WH. Global Health Estimates: Deaths by evaluation.18 However, in existing reports, there is no Cause, Age, Sex and Country, 2000–2012. Geneva: mention about the necessity of training for raters before WHO, 2014, p.9. behavioral tests in animal models of disease. This study 2. Andersen KK, Olsen TS, Dehlendorff C, et al. Hemorrhagic and ischemic strokes compared: Stroke demonstrates the importance of the training process for severity, mortality, and risk factors. Stroke 2009; 40: behavioral tests in animal brain injury models. 2068–2072. 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Re´sume´ L’AVC est une des principales causes de deće`s et d’invalidite´ dans le monde, et son incidence est en augmentation. Pour surmonter le handicap dećoulant d’un AVC, les recherches de translation visant a` de´velopper de nouvelles technologies the´rapeutiques ont e´te´ effectueés et l’occlusion de l’arte`re ce´re´brale moyenne (OACM) chez le rat est le mode`le repre´sentatif. Le re´tablissement apre`s une de´ficience neurologique entraıˆneé par le cerveau endommage´ au niveau des membres controlate´raux constituant le principal objectif du traitement, des controˆles de comportement e´valuant les fonctions pertinentes sont utilise´s. Pour de´terminer l’effet the´rapeutique, un re´sultat fiable d’e´valuation du comportement est une condition preálable. Cependant, les e´tudes sur la fiabilite´ des tests de comportement effectueés sur le mode`le d’occlusion de l’arte`re ce´re´brale moyenne (OACM) chez le rat et la nećessite´ d’une formation preálable n’ont pas encore e´te´ rapporte´s. Dans cette e´tude, les auteurs examinent les fiabilite´s inter-e´valuateur absolues et relatives du score modifie´ de gravite´ neurologique (mNSS), du test du cylindre et du test de marche sur une grille avant la formation, et de nouveau chaque semaine, jusqu’a` ce que la fiabilite´ des re´sultats ait atteint un niveau satisfaisant. La formation comprend l’apprentissage re´pe´te´ du syste`me de notation et la diminution des de´saccords entre les e´valuateurs. Pour la mode´lisation de l’OACM, des rats Sprague-Dawley maˆles adultes ont e´te´ soumis a` une OACM transitoire de 90 minutes. Six e´valuateurs ont effectue´ des controˆles de comportement par l’observation de l’enregistrement videó de rats ayant subi une intervention simuleéetde mode`les d’OACM chez le rat a` 3 ou 7 jours apre`s l’intervention. Un expe´rimentateur inde´pendant a nume´rote´ au hasard chaque clip videó pour que l’expe´rience soit conduite en aveugle. Les re´sultats de fiabilite´se´taient inacceptables avant la formation et se sont ame´liore´s pour atteindre un niveau satisfaisant apre`s 6 semaines de formation a` tous les tests. En conclusion, le mNSS, le test du cylindre et le test de marche sur une grille chez le mode`le d’OACM du rat sont des me´thodes d’e´valuation fiables apre`s avoir effectue´ une formation approprieé.

Abstract Schlaganfa¨lle geho¨ren weltweit zu den ha¨ufigsten Ursachen fu¨r Tod und Invalidita¨t, und ihre Ha¨ufigkeit nimmt zu. Zum Zwecke der Bewa¨ltigung von Behinderungen durch Schlaganfall wurden Translationsforschungsarbeiten zur Entwicklung neuer therapeutischer Technologien durchgefu¨hrt, wobei der Verschluss der mittleren Zerebralarterie (MCAo) bei Ratten das repra¨sentative Modell darstellt. Da die Erholung von neurologischen Beeintra¨chtigungen in den kontralateralen Limbi des gescha¨digten Gehirns das Hauptziel der Behandlung ist, werden Verhaltenstests zur Beurteilung der jeweiligen Funktion eingesetzt. Fu¨r die Bestimmung der therapeutischen Wirkung ist ein zuverla¨ssiges Ergebnis der Verhaltensbewertung Voraussetzung. Berichte zu Studien u¨ber die Zuverla¨ssigkeit von Verhaltenstests im MCAo-Rattenmodell und die Notwendigkeit eines vorherigen Trainings existieren jedoch bis dato nicht. In dieser Studie untersuchten die Autoren relative und absolute Inter-Rater-Reliabilita¨ten modifizierter neurologischer Schweregrade (mNSS), Zylindertests und Gitterlauftests vor dem Training und wiederholten diese jede Woche, bis die Ergebniszuverla¨ssigkeit ein zufriedenstellendes Niveau erreichte. Das Training beinhaltete wiederholtes Erlernen des Bewertungssystems und die Verringerung von Unstimmigkeiten zwischen den Bewertern. Fu¨r die MCAo-Modellierung wurden adulte ma¨nnliche Sprague-Dawley-Ratten 90 Minuten lang transientem MCAo ausgesetzt. Sechs Bewerter fu¨hrten Verhaltenstests durch, indem sie die Videoaufzeichnungen an scheinbetriebenen und MCAo-Rattenmodellen 3 oder 7 Tage nach dem Eingriff beobachteten. Ein unabha¨ngiger Experimentator nahm eine beliebige Nummerierung der einzelnen Videoclips fu¨r Blindversuche vor. Die Ergebniszuverla¨ssigkeit war vor dem Training inakzeptabel und verbes- serte sich nach 6 Wochen Training in allen Tests auf ein zufriedenstellendes Niveau. Zusammenfassend la¨sst sich sagen, dass mNSS, Zylindertest und Gitterlauftest mit dem MCAo-Rattenmodell nach Durchfu¨hrung eines entsprechenden Trainings zuverla¨ssige Bewertungsmethoden sind. 490 Laboratory Animals 53(5)

Resumen El infarto es una de las principales causas de muerte y discapacidad en todo el mundo, y su incidencia esta´ aumentando. Para sobrepasar el deterioro por infarto, se han realizado distintos estudios translacionales para desarrollar nueva tecnologıá terapeútica y la oclusioń de la arteria cerebral media (MCAo) en ratas es el modelo de representacioń. Ya que la recuperacioń del deterioro neurolo´gico de las extremidades contral- aterales del cerebro danãdo es el principal objetivo del tratamiento, se utilizan pruebas de comportamiento que evaluán la funcioń relevante. Para determinar el efecto terapeútico, un resultado fiable de la evaluacioń del comportamiento es esencial. Sin embargo, todavıá no se han obtenido resultados de estudios sobre la fiabilidad de las pruebas de comportamiento en modelos de ratas con MCAo y la necesidad de formacioń previa. En este estudio, los autores investigan fiabilidades relativas y absolutas entre codificadores de la puntua- cioń de la gravedad neurolo´gica modificada (mNSS), pruebas de cilindros y pruebas de caminar en rejillas antes de la formacioń y despue´s cada semana hasta que los resultados de la fiabilidad alcancen unos niveles satisfactorios. La formacioń incluye un aprendizaje repetido del sistema de puntuacioń y unos desacuerdos en disminucioń entre los codificadores. Para el modelo con MCAo, distintas ratas macho Sprague-Dawley fueron sometidas a 90 minutos de MCAo transitoria. Seis codificadores llevaron a cabo pruebas de comportamiento a trave´s de la observacioń de grabaciones en vı´deo de ratas MCAo y con simulacro a los 3 o 7 dıás despue´sdela intervencioń. Un experimentador independiente numero´ aleatoriamente cada videoclip para un experimento ciego. Los resultados de las fiabilidades fueron inaceptables antes de la formacioń y mejoraron hasta llegar a un nivel satisfactorio despue´s de 6 semanas de formacioń en todas las pruebas. En conclusioń, mNSS, las pruebas de cilindro y la prueba de andar en rejilla con el modelo de rata con MCAo son me´todos fiables de evaluacioń despue´s de llevar a cabo una formacioń apropiada. Original Article Laboratory Animals 2019, Vol. 53(5) 491–499 ! The Author(s) 2018 Effect of three opioid-based analgesic Article reuse guidelines: sagepub.com/journals- protocols on the perioperative permissions DOI: 10.1177/0023677218815203 autonomic-mediated cardiovascular journals.sagepub.com/home/lan response in sheep

Luca Bellini1 and Giulia Maria De Benedictis2

Abstract Few reports evaluate the clinical effects of opioids in sheep during experimental surgical procedures. Catecholamine-mediated haemodynamic changes resulting from surgical noxious stimulation are blunted by opioids. The aim of this study was to evaluate the efficacy of three opioid-based analgesic protocols in avoiding a 20% increase in heart rate (HR) and/or mean arterial blood pressure (MAP) during experimental intervertebral disk nucleotomy in sheep. Eighteen female Brogna sheep were anaesthetized with propofol and maintained with a fixed end-tidal isoflurane concentration of 1.5 Æ 0.1%. Sheep were assigned to one of three groups that intravenously received methadone 0.3 mg/kg (group M), fentanyl 2 mg/kg followed by 10 mg/kg/h (group F), or buprenorphine 10 mg/kg and 30 minutes later ketamine 1 mg/kg followed by 5 mg/kg/h (group BK). Intravenous fentanyl at 2 mg/kg would have been used for rescue analgesia in case HR and/or MAP had increased. During surgery, HR and MAP values did not increase over 20% in all groups. All animals maintained the percentage change between -4 and 7% for both variables; only one sheep in group BK had an increase in MAP superior to 20% after ketamine administration before surgical stimulation. In group M, HR decreased over time and in group BK, MAP tended to increase during surgery. All the opioid-based protocols tested were able to control the cardiovascular response to noxious stimulation in sheep undergoing spinal surgery, although ketamine may have represented a confounding factor.

Keywords sheep, methadone, fentanyl, buprenorphine, analgesia, spinal surgery

Date received: 3 July 2018; accepted: 27 October 2018

Introduction lower efficacy than fentanyl patch at 2 mg/kg/h in relieving postoperative pain after unilateral tibial osteotomy Sheep are commonly used as in vivo model for experi- in sheep.6 mental orthopaedic procedures involving spinal or long- Fentanyl 10 mg/kg/h was shown to decrease end-tidal bone surgeries.1–8 Analgesic protocols that include isoflurane requirement by 22.6%, compared to the con- opioids are now more common during this type of sur- trol group, in sheep undergoing orthopaedic surgery, gery and among those compounds, buprenorphine is the and recovery quality was scored as good to excellent most widely used.1 Buprenorphine at 6 mg/kg administered intravenously (IV) was seen to increase the thermal 1Veterinary Teaching Hospital, University of Padua, Italy threshold in sheep, although the same dose caused no 2Department of Animal Medicine, Production and Health, change in response to mechanical stimuli.9,10 More University of Padua, Italy recently, there has been renewed interest in the systemic Corresponding author: use of opioids other than buprenorphine, such as metha- 5,6,7,11–14 Luca Bellini, Veterinary Teaching Hospital, University of Padua, done and fentanyl, in sheep. One study observed Viale dell’Universita` 16, 35020 Legnaro, PD, Italy. that intramuscular (IM) buprenorphine 10 mg/kg had a Email: [email protected] 492 Laboratory Animals 53(5) in most of the animals.5 Methadone 0.5 mg/kg injected Subcutaneous (SQ) ivermectin (Ivomec ovine, IV was associated with dexmedetomidine or xylazine in Merial Italia Spa, Milan, Italy) 200 mg/kg was adminis- sheep, and its effects on sedation and some cardiovas- tered as an endoparasite treatment at least 2 weeks cular variables were similar to those observed after the before the procedures. administration of the alpha-2 agonist alone.11,12 Sheep were housed in pens containing six animals Another study used methadone as a premedication each, and fed with a commercial pellet (Compli agent in combination with midazolam, before right Sheep, Tecnozoo, Piombino Dese, Padua, Italy) and heart bypass in sheep.15 Nevertheless, none of those hay diet. Prior to the experimental procedure, clinical studies evaluated the analgesic effects of methadone. examination, complete blood count and serum chemis- Roizen and colleagues observed in human patients try were performed, on the basis of which sheep were that skin incision caused a release of noradrenaline, and considered healthy. this may be reduced dose-dependently by volatile Sheep were evenly distributed between three groups anaesthetic or by the concurrent administration of mor- on the basis of the IV analgesic protocol used: group phine.16 A lower increase in arterial blood pressure due M, 0.3 mg/kg of methadone (Comfortan, Dechra, to skin incision was obtained at higher alveolar concen- Turin, Italy); group F, fentanyl (Fentadon, Dechra, tration of the anaesthetic or with incremental doses of Turin, Italy) 2 mg/kg followed by 10 mg/kg/h; and morphine. Sympathetic-induced changes in heart rate group BK, ketamine (Ketavet 100, Intervet (HR) and mean arterial blood pressure (MAP), propor- Productions Srl, Aprilia, Italy) 1 mg/kg followed by tional to the surgical stimulation, have been demon- 5 mg/kg/h and buprenorphine (Buprenodale, Dechra, strated in human patients undergoing abdominal Turin, Italy) 0.01 mg/kg. Sheep were weighed the morn- surgical procedures.17 Also in isoflurane-anaesthetized ing of the day scheduled for the procedure. sheep undergoing experimental tibial surgery, clinically significant increases in HR and MAP were observed Materials and methods during intense surgical stimuli.3 In that study, an increase in HR or MAP between 13 and 30% was asso- Food, but not water, was withheld for 12 hours before ciated with an electroencephalographic pattern compat- the experiment. Medetomidine (Sedator; Dechra, ible with arousal or paradoxical arousal during noxious Turin, Italy) 8 mg/kg was administered IV in the jugular stimulation. vein. Ten minutes later, sheep were moved to the pre- To our knowledge, no study has so far assessed the surgery area, where the marginal auricular vein and the ability of different opioid-based protocols to minimize auricular artery were cannulated with a 20 gauge over- cardiovascular changes due to surgical stimulation in the-needle IV catheter. After 30 minutes, anaesthesia sheep. The aim of this study was to evaluate the efficacy was induced IV with propofol (Propofol Kabi; of three different opioid-based analgesic protocols in Fresenius Kabi Italia s.r.l., Italy) 4 mg/kg. Airways preventing increases in HR and/or invasive MAP in were secured with endotracheal tubes, and the animals isoflurane-anaesthetized sheep undergoing experimen- were connected to a circle breathing system and mech- tal spinal surgery. We hypothesized that all the treat- anically ventilated in a pressure control mode (Datex- ments evaluated might prevent a 20% increase in HR Ohmeda 7900 SmartVent; GE Healthcare, Finland), at and/or invasive MAP, considered as indirect clinical a peak pressure of 15 cmH2O and a respiratory rate signs of sympathetic activation induced by surgical adjusted to maintain normocapnia between 37 and stimulation. 45 mmHg. Fresh gas flow was set to 4 L/minute and after 10 minutes decreased to 1 L/minute, with a frac- Animals tion of inspired oxygen of 0.5. Sheep were positioned in sternal recumbency on a heating pad, and a gastric tube Eighteen Brogna, non-pregnant female sheep were was inserted through their mouth in order to minimize enrolled in a prospective controlled study and under- rumen distension. An isoflurane target end-tidal con- went spinal surgery consisting in a transpedicular inter- centration (FE’Iso; IsoFlo; Zoetis, Rome, Italy) of vertebral disk nucleotomy from L1 to L4–L5 as part of 1.5 0.1% was set and maintained throughout the pro- another experimental study. Sheep weighed 47 8kg cedure. A multiparameter monitor (Datex S/5; GE and were 3 years old. Healthcare, Helsinki, Finland) was used to record The study was performed with the approval of the HR, invasive arterial blood pressure, respiratory rate, Animal-welfare Body of the University of Padova haemoglobin oxygen saturation and end-tidal gas (OPBA Authorization 7/2014) and the Italian analysis. Ministry of Health, according to European (Directive After 15 minutes of stable target FE’Iso, sheep 2010/63/EU) and Italian regulations (Legislative received one of the three treatments. All drugs were Decree 26/2014). injected IV over 2 minutes, at least 10 minutes before Bellini and De Benedictis 493 skin incision, while buprenorphine was injected at least 0.8 and of 0.05. Data were analysed using 30 minutes before incision. Ketamine was infused for GraphPad Prism 6.0 (GraphPad software, CA, US) no longer than 2 hours; Ringer’s lactate solution at and P < 0.05 was considered statistically significant. 5 ml/kg/h was infused during the entire procedure. Three surgical periods were identified: from skin inci- Results sion to complete spine exposure, which was defined as Tspine; the nucleotomy period, called Tnucleo; and the All sheep completed the study without complications period from the beginning of tissue reconstruction to during surgery or the postoperative period; the proced- the end of the skin suture, defined Tsuture. ure lasted between 125 and 270 minutes. Rescue anal- During surgery, an increase in HR and/or invasive gesia with fentanyl was never required and the target MAP of more than 20%, compared to the value mea- concentration of volatile anaesthetic was achieved in all sured 5 minutes earlier and lasting more than 30 sec- the animals. Figure 1 shows the percentage change of onds, was considered indicative of inadequate HR and MAP in each surgical time point considered, intraoperative analgesia.2, 13 In such a case, in order for every single group. At skin incision, HRs in group to provide rescue analgesia, the study design established M decreased more than 20% in two animals, while in that fentanyl at 2 mg/kg had to be administered IV and group F and group BK, they minimally changed after that the same dose had to be repeated at 5 minute inter- opioid administration (Figure 1). Six animals in group vals, until the parameters would have returned to pre- M and group F, and five sheep in group BK, remained vious values. At the end of the procedure, after the below the target limit for MAP (20% increase) at all return of spontaneous ventilation, the sheep were dis- time points (Figure 1). One animal in group BK had an connected from the breathing system and positioned in increase of 46% in MAP immediately after ketamine sternal recumbency with the head elevated. Once the loading dose administration, although the changes animals were able to support their head and vigorous observed during the rest of the procedure remained swallows were observed, the endotracheal tube was below 20% (Figure 1). removed, and the animals were returned to their pen Moreover, in group M, HR decreased over time and and monitored for behavioural signs of pain or abnor- differed statistically from Tpre at Tskin,Tspine and Tsuture mal posture. Amoxicilline-clavulanate (Synulox; Zoetis, (Figure 2(a)). In group F and group BK, cardiac fre- Rome, Italy; 25 mg/kg, twice daily, SQ) was adminis- quency remained constant throughout the procedure: tered on the day of the surgery and repeated for the no statistical differences were observed between Tpre first 7 days after surgery. Carprofen (Rimadyl; Zoetis, and other time points (Figure 2(b) and (c)). Rome, Italy; 3 mg/kg once a day, SQ) and tramadol In group M, MAP tended to decrease throughout (Altadol; Formevet Spa, Milan, Italy; 4 mg/kg, twice the anaesthesia and values measured at Tsuture were daily, IM) were administered at the end of surgery and statistically different from those at Tpre (Figure 2(d)). continued for the following 3 days, or longer if needed. Group F showed no statistical differences in MAP (Figure 2(e)). Blood pressure increased in group BK Statistical analysis from Tpre over time, with statistically significant difference at Tspine and Tnucleo (Figure 2(f)). Percentage change in HR and MAP was expressed as Oesophageal temperature remained between 36.9 median (min-max) and calculated every 5 minutes as: and 38.0C with no differences in mean values among 100 (Xt Xt-5)/Xt-5. Normally distributed data were groups (P ¼ 0.397). expressed as mean SD, otherwise as median (min- Recovery was smooth in all animals and no differ- max). The anaesthesia time points considered for the ences in extubation time (time between disconnection analysis were: Tpre, which includes values recorded for from the anaesthetic machine and removal of the endo- 15 minutes of stabilization after having reached target tracheal tube) were observed among groups, resulting FE’Iso; Tadmin, which includes values recorded 5 min- in 9 3, 11 4 and 12 5 minutes in groups M, F and utes after starting drug administration; Tskin, that BK, respectively (P ¼ 0.396). All sheep were able to includes measures obtained 5 minutes after skin inci- stand and walk within 35 minutes after extubation. sion; Tspine;Tnucleo; and Tsuture. Variations of HR and MAP over time were evaluated within each group with Discussion one-way analysis of variance. A Tukey’s multiple comparisons test was used to detect statistical difference Among indirect intraoperative indicators of nocicep- between Tpre and the other time points considered. tion, changes over time in HR and arterial blood pres- Based on the results of a similar study,2 sample size sure are commonly used.2,3,16–20 A sudden increase of was calculated to obtain a mean difference of more than 20% in these parameters suggested a poor 20% 10% compared to baseline, with a power of analgesic coverage during orthopaedic surgery in 494 Laboratory Animals 53(5)

Figure 1. Percentage change in heart rate and invasive mean arterial blood pressure at different procedure time points in isoflurane-anaesthetized sheep undergoing transpedicular intervertebral disk nucleotomy. Animals intravenously received methadone 0.3 mg/kg (group M), fentanyl 2 mg/kg followed by 10 mg/kg/h (group F) or ketamine 1 mg/kg followed by 5 mg/kg/h co-administered with buprenorphine 0.01 mg/kg (group BK) for intraoperative analgesia. Data are reported as medians, interquartile ranges, and minimum and maximum values. Anaesthesia time points reported on the x-axis are: Tpre, measurements recorded for 15 minutes of stabilization after having reached target FE’Iso; Tadmin, values recorded 5 minutes after starting drug administration; Tskin, which includes measures obtained 5 minutes after skin incision; Tspine, from skin incision to complete spine exposure; Tnucleo, from spine exposure to the end of nucleotomy; and Tsuture, from tissue reconstruction to the end of the skin suture. sheep.3 The opioid-based protocols evaluated in our 10 mg/kg/h, usually preceded by a loading dose of study maintained HR and/or MAP percentage change 5 mg/kg,5,13 but no detailed information is available inferior to 20%. on the intraoperative cardiovascular response to nox- The cardiovascular response to noxious stimulation ious stimulation at these doses. In sheep receiving fen- after systemic methadone administration has not been tanyl at 10 mg/kg/h during orthopaedic surgery, a studied in sheep, despite its use in painful experimental progressive decrease in isoflurane consumption from procedures in this species.15 The doses reported in the 1.5 to 0.9% was required to keep HR and MAP con- literature for methadone range from 0.1 to 0.5 mg/kg stant over the anaesthetic period.5 In our study, for IV, intramuscular or epidural routes, and the although the end-tidal concentration of isoflurane and impact of this opioid on HR seems to be negli- the fentanyl infusion rate were not modified, those par- gible.11,12,14,15,21 Methadone at a dose of 0.3 mg/kg ameters did not change, probably because the surgical administered by epidural route provided more than noxious stimulation did not vary over time. 210 minutes of analgesia without signs of bradycar- Two studies have clinically evaluated the effect of dia.21 In awake sheep, IV administration of 0.5 mg/kg transdermal fentanyl patch at a drug-releasing rate of methadone and acepromazine caused sedation that 2.0-2.6 mg/kg/h in sheep undergoing lumbar interverte- lasted 60 minutes without affecting HR.14 The same bral spinal fusion or unilateral left tibial osteotomy, but dose of methadone, co-administered with xylazine or the evaluation of the analgesic efficacy was limited to dexmedetomidine, caused bradycardia, which was not the postoperative period.6,7 Although both trials used different from what observed with the alpha-2 agonist isoflurane for the maintenance of anaesthesia, the mean alone.11,12 In our study, methadone administered to end-tidal concentration delivered was reported only in sheep at 0.3 mg/kg was able to minimize the percentage one study. Ahern and co-workers reported a mean increases in HR and MAP during surgery, thus suggest- value of isoflurane end-tidal concentration of 2.2% at ing an adequate control of the cardiovascular response the end of the tibial osteotomy.6 In our study, the par- secondary to surgical stimuli. Nevertheless, a decrease tial pressure of isoflurane in the expired gases was in HR was observed after methadone administration maintained at 1.5 0.1%, which represents the mini- and this might be attributed to the vagally mediated mum alveolar concentration (MAC) for this species. effect, as reported in anaesthetized dogs receiving a The lower isoflurane consumption reported in our full -opioid receptor agonist.22 study, compared to that observed by Ahern and col- Fentanyl infusion has been used for intraoperative leagues, might be explained by the different fentanyl analgesia in sheep undergoing major surgery by either infusion rate used, although differences in the intensity IV or transdermal administration routes. IV infusion of noxious stimulation should be also considered. rates reported in different studies vary from 2 to Moreover, transdermal absorption during anaesthesia Bellini and De Benedictis 495

Figure 2. Mean SD of intraoperative heart rate (on the left) and invasive mean arterial blood pressure (on the right), measured at different procedure time points in isoflurane-anaesthetized sheep undergoing transpedicular intervertebral disk nucleotomy that intravenously received methadone 0.3 mg/kg ((a) and (d)), fentanyl 2 mg/kg followed by 10 mg/kg/h ((b) and (e)) or ketamine 1 mg/kg followed by 5 mg/kg/h co-administered with buprenorphine 0.01 mg/kg ((c) and (f)) as intraoperative analgesia. Anaesthesia time points reported on the x-axis are: Tpre, measurements recorded for 15 minutes of stabilization after having reached target FE’Iso; Tadmin, values recorded 5 minutes after starting drug administration; Tskin, that includes measures obtained 5 minutes after skin incision; Tspine, from skin incision to complete spine exposure; Tnucleo, from spine exposure to the end of nucleotomy; and Tsuture, from tissue reconstruction to the end of the skin suture. *P < 0.05 from Tpre. may be influenced by several factors that could have different infusion times of 150 and 100 minutes, respect- affected fentanyl plasma concentration in Ahern’s ively.5 Furthermore, as previously reported, recovery study.6 from anaesthesia was smooth without any signs of Mean extubation time was similar between our study excitement or sedation recurrence.5,7 This may support and that observed by Funes and co-workers, despite the hypothesis that fentanyl in sheep does not 496 Laboratory Animals 53(5) accumulate significantly after an infusion lasting noxious stimulus. In sheep undergoing experimental between 2 and 3 hours, as it was demonstrated to do tibial osteotomy, an intramuscular dose of 10 mg/kg in dogs after 4 hours of fentanyl infusion at 10 mg/kg/ caused a decrease in HR that was reversed by surgical h.23 stimulation 40 minutes after its injection.8 In the same Buprenorphine is considered a partial agonist of study, MAP was not influenced by medetomidine -opioid receptor and, when compared to the full agon- administration. Considering that, in our study, a low ist fentanyl, it is less adequate for the management of dose of medetomidine was used and the surgery began postoperative orthopaedic pain in sheep.6 Analgesic 80 minutes after its injection, an effect of the alpha-2 activity of IV buprenorphine 0.01 mg/kg has been eval- agonist on the cardiovascular system seems to be unli- uated in sheep by mechanical and thermal threshold kely. Propofol and isoflurane may have contributed to tests, and a dose-dependent decrease in response was suppress the response to noxious stimulation, although measured only for the thermal threshold test.9,10 they should have influenced all the animals in the same Because of the conflicting results on nociceptive thresh- way, as the doses used were similar. The lack of any old tests and the elevated concentration of isoflurane evident sympathetically mediated cardiovascular reported by Ahern and co-workers during orthopaedic response to surgery may also be due to a mild surgical surgery,6 buprenorphine at 0.01 mg/kg was expected to stimulation, which could be less intense than expected. provide poor analgesia for intervertebral disk nucleot- The use of a control group undergoing the same surgi- omy. There is recognized evidence that subanaesthetic cal procedure without analgesics could have certainly doses of ketamine provide analgesia, preventing hyper- provided evidence of the noxious stimulation intensity algesia and blunting central sensitization, when they are and the effects of the three protocols, but such a con- used as adjuvant in analgesic protocols, both in humans dition would have been considered unethical; conse- and animals.24 An infusion of ketamine at a subanaes- quently, it was not contemplated. Indeed, this thetic dose (10 mg/kg/minute) was thus co-administered opportunistic study was part of a long-term ortho- in our study to improve nociception control. The same paedic experimental trial, and untreated intraoperative rate of ketamine infusion, associated with lidocaine at nociception is known to be detrimental with regard to 20 mg/kg/minute, was used as adjuvant in isoflurane- animal welfare, and is potentially associated with post- anaesthetized sheep undergoing meniscal surgery.4 In operative distress and complications. In addition, in the same study, fentanyl at 2 mg/kg/h was provided by view of the 3 R principles (replacement, reduction and transcutaneous administration as intraoperative anal- refinement), collecting both surgical and anaesthetic gesia. Compared to the control group, which received information from the same animal allows the reduction only a transdermal fentanyl patch, isoflurane require- of the number of animals involved and refinement of ment was about 23% less when ketamine and lidocaine the analgesic approach. were used. In group BK, no animals showed a HR per- Another limitation is the use of cardiovascular vari- centage increase superior to 20%, whereas only one ables as the sole sign of intraoperative nociception. In sheep had an increase of more than 20% in MAP isoflurane-anaesthetized sheep undergoing tibial osteot- immediately after the administration of ketamine omy, electroencephalographic analysis was evaluated bolus. This increase can be considered an effect of keta- beside HR and systemic arterial blood pressure as an mine injection since, at that time, no noxious stimula- indicator of intraoperative nociception. However, the tion had been applied. Moreover, before ketamine results of that study showed that haemodynamic bolus, no signs of abnormal cardiovascular parameters changes were more reliable and correlated with the had been noted. Similar to what we have observed in intensity of intraoperative nociceptive stimulation sheep, dogs under isoflurane anaesthesia receiving keta- better than the electroencephalographic pattern.3 mine infusion at different plasma concentration showed Moreover, to the authors’ knowledge, there are not an increase in cardiovascular parameters.20 The cardio- objective methods for the acquisition of real-time infor- vascular response to ketamine might have been second- mation to assess surgical nociception in anaesthetized ary to a central sympathomimetic effect that this drug sheep. seems to cause even at subanaesthtic doses in sheep and 20-24 dogs, but not in humans. Conclusion This study has some limitations. First, the concurrent administration of sedatives and anaesthetics may All the opioid-based protocols evaluated were able to have affected the cardiovascular response secondary to minimize the cardiovascular response to noxious surgi- the noxious surgical stimulation. Medetomidine may cal stimulation in isoflurane-anaesthetized sheep. This attenuate the sympathetic activation due to surgery, study indicates that, with the tested protocols, HR and and its effect is influenced by the dose, and the time MAP do not change by over 20% during the surgical between administration and the application of the stimulation. The cardiovascular activation caused by Bellini and De Benedictis 497 ketamine may represent a confounding factor, and this 8. Ka¨stner SB, Kutter AP, von Rechenberg B, et al. drug should be administered before the application of Comparison of two pre-anaesthetic medetomidine doses noxious stimuli if the sympathetic response to surgery in isoflurane anaesthetized sheep. Vet Anaesth Analg has to be monitored. Further studies are warranted in 2006; 33: 8–16. order to evaluate the analgesic efficacy of these proto- 9. Nolan A, Livingston A and Waterman AE. Investigation of the antinociceptive activity of buprenorphine in sheep. cols in the early postoperative period. Br J Pharmacol 1987; 92: 527–533. 10. Waterman AE, Livingston A and Amin A. Further stu- Acknowledgements dies on the antinociceptive activity and respiratory effects The results of this investigation were presented at the of buprenorphine in sheep. J Vet Pharmacol Ther 1991; Association of Veterinary Anaesthetists autumn meeting 14: 230–234. 2017, and are published as abstract in the proceedings of the 11. Borges LP, Nishimura LT, Carvalho LL, et al. conference. The authors would like to thank Dr Sara Bordin Behavioral and cardiopulmonary effects of dexmedetomi- for proofreading assistance and constructive comments. dine alone and in combination with butorphanol, methadone, morphine or tramadol in conscious sheep. Vet Declaration of Conflicting Interests Anaesth Analg 2016; 43: 549–560. 12. de Carvalho LL, Nishimura LT, Borges LP, et al. The author(s) declared no potential conflicts of interest with Sedative and cardiopulmonary effects of xylazine alone respect to the research, authorship and/or publication of this or in combination with methadone, morphine or trama- article dol in sheep. Vet Anaesth Analg 2016; 43: 179–188. 13. Barletta M, Kleine SA, Hofmeister EH, et al. Funding Determination of the minimum alveolar concentration The author(s) received no financial support for the research, of isoflurane that blunts adrenergic responses in sheep authorship and/or publication of this article. and evaluation of the effects of fentanyl. Am J Vet Res 2016; 77: 119–126. ORCID iD 14. Nishimura LT, Villela IOJ, Carvalho LL, et al. The effect of acepromazine alone or in combination with metha- Luca Bellini http://orcid.org/0000-0002-7145-6115 done, morphine, or tramadol on sedation and selected Giulia Maria De Benedictis http://orcid.org/0000-0002- cardiopulmonary variables in sheep. Vet Med Int 2017; 8422-6829 2017: 7507616. 15. Schauvliege S, Narine K, Bouchez S, et al. Refined anaes- References thesia for implantation of engineered experimental aortic 1. Coulter CA, Flecknell PA and Richardson CA. Reported valves in the pulmonary artery using a right heart bypass analgesic administration to rabbits, pigs, sheep, dogs and in sheep. Lab Anim 2006; 40: 341–352. non-human primates undergoing experimental surgical 16. Roizen MF, Horrigan RW and Frazer BM. Anesthetic procedures. Lab Anim 2009; 43: 232–238. doses blocking adrenergic (stress) and cardiovascular 2. De Benedictis GM, Giorgi M, Depase A, et al. responses to incision-MAC BAR. Anesthesiology 1981; Cardiovascular effects and intraoperative pharmacokinet- 54: 390–398. ics of tramadol in sheep undergoing spinal surgery. Vet 17. Schubert A, Palazzolo JA, Brum JM, et al. Heart rate, Anaesth Analg 2017; 44: 1245–1252. heart rate variability, and blood pressure during peri- 3. Otto KA and Mally P. 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22. Laubie M, Schmitt H and Vincent M. Vagal bradycardia injection and constant rate infusion in dogs. Vet produced by microinjections of morphine-like drugs into Anaesth Analg 2006; 33: 266–273. the nucleus ambiguus in anaesthetized dogs. Eur J 24. Gorlin AW, Rosenfeld DM and Ramakrishna H. Pharmacol 1979; 59: 287–291. Intravenous sub-anesthetic ketamine for perioperative 23. Sano T, Nishimura R, Kanazawa H, et al. analgesia. J Anaesthesiol Clin Pharmacol 2016; 32: Pharmacokinetics of fentanyl after single intravenous 160–167.

Re´sume´ Peu de rapports e´valuent les effets cliniques des opioı¨des chez les ovins lors des interventions chirurgicales expe´rimentales. Les modifications he´modynamiques me´dieés par les catećholamines re´sultant d’une stimulation chirurgicale nocive sont brideés par les opioı¨des. Le but de cette e´tude e´tait d’e´valuer l’efficacite´ de trois protocoles analge´siques a` base d’opioı¨des pour e´viter une augmentation de 20 % de la fre´quence cardiaque (FC) et/ou de la pression arte´rielle moyenne (PAM) au cours d’une nucleótomie experimentale de disque interverte´bral percutaneé chez les ovins. Dix-huit brebis Brogna ont e´te´ anesthe´sieés au propofol et maintenues en se´dation par une concentration d’isoflurane fixe en fin d’expiration de 1,5 0,1 %. Les brebis ont e´te´ affecteés a` l’un des trois groupes auxquels a e´te´ administre´, par voie intraveineuse: de la me´thadone a` 0,3 mg/kg (groupe M); du fentanyl a` 2 mg/kg suivi de 10 mg/kg/h (groupe F); de la bupreńorphine a` 10 mg/kg et 30 minutes plus tard de la ke´tamine a` 1 mg/kg suivie de 5 mg/kg/h (groupe BK). Le fentanyl par voie intraveineuse a` 2 mg/kg aurait e´te´ utilise´ pour l’analge´sie de sauvetage en cas d’augmentation de la FC et/ou de la PAM. Au cours de la chirurgie, les valeurs de FC et de PAM n’ont pas augmente´ de plus de 20 % dans tous les groupes. Tous les animaux ont maintenu une variation de pourcentage situeé entre -4 % et 7 % pour les deux variables; seul une brebis du groupe BK a pre´sente´ une augmentation de la PAM supe´rieure a` 20 % apre`s l’administration de la ke´tamine preće´dant la stimulation chirurgicale. Dans le groupe M, la FC a diminue´ au fil du temps et dans le groupe BK, la PAM a eu tendance a` augmenter au cours de la chirurgie. Tous les protocoles a` base d’opioı¨des teste´s ont e´te´ en mesure de controˆler la re´ponse cardiovasculaire a` la stimulation nocive chez les ovins subissant une chirurgie rachidienne, bien que la ke´tamine ait pu repre´senter un facteur de confusion.

Abstract Nur wenige Berichte bewerten die klinischen Auswirkungen von Opioiden bei Schafen wa¨hrend experimen- teller chirurgischer Eingriffe. Katecholaminvermittelte ha¨modynamische Vera¨nderungen, in Folge scha¨dli- cher chirurgischer Stimulation werden durch Opioide kontrolliert. Ziel dieser Studie war es, die Wirksamkeit von drei opioidbasierten Analgetika-Protokollen zu bewerten, um einen Anstieg der Herzfrequenz (HR) und/ oder des mittleren arteriellen Blutdrucks (MAP) um 20 % wa¨hrend der experimentellen Bandscheiben- Nukleotomie beim Schaf zu vermeiden. Achtzehn weibliche Brogna-Schafe wurden mit Propofol beta¨ubt und bei einer konstanten end-tidalen Isoflurankonzentration von 1,5 0,1 % gehalten. Die Schafe wurden einer von drei Gruppen zugeordnet, die intraveno¨s Folgendes erhielten: Methadon 0,3 mg/kg (Gruppe M); Fentanyl 2 mg/kg gefolgt von 10 mg/kg/h (Gruppe F); Buprenorphin 10 mg/kg und 30 Minuten spa¨ter Ketamin 1 mg/kg gefolgt von 5 mg/kg/h (Gruppe BK). Intraveno¨se Fentanylgabe von 2 mg/kg wa¨re als Notfall-Analgesie eingesetzt worden, falls eine Erho¨hung von HR und/oder MAP aufgetreten wa¨re. Wa¨hrend der Operation stiegen die HR- und MAP-Werte in keiner der Gruppen um mehr als 20 %. Alle Tiere bewegten sich fu¨r beide Variablen innerhalb der prozentualen Vera¨nderung von -4 % und 7 %; nur ein Schaf in der Gruppe BK hatte einen Anstieg des MAP von u¨ber 20 % nach Ketaminverabreichung vor der chirurgischen Stimulation. In der Gruppe M nahm die HR im Laufe der Zeit ab, und in der Gruppe BK nahm der MAP wa¨hrend der Operation tendenziell zu. Mit allen getesteten opioidbasierten Protokollen konnte die kardiovaskula¨re Reaktion auf die scha¨dliche Stimulation bei Schafen wa¨hrend der Wirbelsa¨ulenoperation kontrolliert werden, obwohl Ketamin ein Sto¨rfaktor gewesen sein ko¨nnte.

Resumen Son pocos los estudios que evaluán los efectos clıńicos de los opioides en ovejas durante procedimientos quiru´rgicos de experimentacioń. Los cambios hemodina´micos mediante catecolamina resultantes de la Bellini and De Benedictis 499 estimulacioń quiru´rgica nociva se ven atenuados por los opioides. El objetivo de este estudio era evaluar la eficacia de tres protocolos de analge´sicos basados en opioides para evitar un aumento del 20% del ritmo cardıáco (HR) y/o la presioń sanguıńea arterial media (MAP) durante la nucleotomıá de disco intervertebral de experimentacioń con ovejas. Dieciocho ovejas Brogna hembra fueron anestesiadas con propofol y fueron mantenidas con una concentracioń de isoflurano expirada final fija de 1,5 0,1%. Las ovejas fueron asignadas a uno de tres grupos que recibieron de forma intravenosa: metadona 0,3 mg/kg (grupo M); fentanilo 2 mg/kg seguido de 10 mg/kg/h (grupo F); buprenorfina 10 mg/kg y 30 minutos despue´s ketamina 1 mg/kg seguido de 5 mg/kg/h (grupo BK). El fentanilo intravenoso a 2 mg/kg se hubiera utilizado para analgesia de rescate en caso de aumento del HR y/o MAP. Durante la cirugıá, los valores de HR y MAP no aumentaron por encima del 20% en ninguń grupo. Todos los animales mantuvieron el cambio de porcentaje entre el -4% y el 7% para ambas variables; uńicamente una oveja del grupo BK tuvo un aumento del MAP superior al 20% despue´sde una administracioń de ketamina antes de la estimulacioń quiru´rgica. En el grupo M, el HR descendio´ con el tiempo y en el grupo BK, el MAP tendio´ a subir durante la cirugıá. Todos los protocolos basados en opioides probados pudieron controlar la respuesta cardiovascular frente a la estimulacioń nociva en ovejas sometidas una cirugıá espinal, aunque la ketamina puede haber representado un factor desconcertante. Original Article Laboratory Animals 2019, Vol. 53(5) 500–507 ! The Author(s) 2018 A REDCap application that links Article reuse guidelines: sagepub.com/journals- researchers, animal facility staff permissions DOI: 10.1177/0023677218815723 and members of the IACUC in journals.sagepub.com/home/lan animal health monitoring

Cristobal Carvajal1, Catalina Vallejos2, Dominique Lemaitre3, Jorge Ruiz2, Camila Guzmań4, Valentina Aguilera4, Diego Banõ5 and Sebastiań D. Calligaris6

Abstract Research studies involving animal experimentation are regulated by the Institutional Animal Care and Use Committee (IACUC). To this end, the IACUC must integrate the information provided by the investigators of each preclinical study and the veterinarians from the animal facility in order to monitor and approve the process. Using a paper-based system to collect animal health and welfare data is a common, albeit time- consuming practice, prone to transcription and reading errors, not to mention inconvenient for veterinarians and investigators wishing to make timely and collaborative decisions when animal welfare is at risk. We created a web-based monitoring system focused on animal health with the potential to improve animal welfare. The data management system is based on REDCap software, which enables data integration in order to offer a solution for animal welfare assessment. The proposed scheme includes key indicators of general health status, such as environment, physical/nutritional information, and behavioral parameters during animal breeding and experimentation, as important components of animal welfare. In addition, the system facilitates communication of this information among researchers, animal facility staff, and the IACUC. REDCap is available to non-profit organizations, and may be adapted and replicated by institutions interested in and responsible for animal care, and used in research. REDCap is an excellent tool for promoting good practices that benefit experimental animal health.

Keywords REDCap, web-based system, animal health, animal welfare and ethics

Date received: 21 March 2018; accepted: 5 November 2018

1Instituto de Ciencias e Innovacioń en Medicina, Clıńica Alemana Facultad de Medicina, Clinica Alemana, Universidad del Introduction Desarrollo, Chile 2Laboratory Animal Facility, Facultad de Medicina Clıńica Alemana, Animal ethics guidelines provide that institutions inter- Universidad del Desarrollo, Chile ested in using animals for research and teaching must 3Centro de Fisiologıá Celular e Integrativa, Facultad de Medicina, appoint an Institutional Animal Care and Use Clıńica Alemana Universidad del Desarrollo, Santiago, Chile 4 Committee (IACUC) or equivalent oversight body to TecnologıáMe´dica, Facultad de Medicina Clıńica Alemana, Universidad del Desarrollo, Chile oversee laboratory animal welfare, according to the 5Facultad de Ciencias Fı´sicas y Matema´ticas, Universidad de Chile, animal care and use program established by each insti- Chile 1 tution. The IACUC is responsible for the review, 6Centro de Medicina Regenerativa Facultad de Medicina Clıńica approval, and monitoring of projects that include Alemana, Universidad del Desarrollo, Chile animal experimentation, and for the periodic inspection Corresponding author: of animal facilities, animal care programs, and concerns Sebastiań D. Calligaris, PhD, Centro de Medicina Regenerativa involving the care and use of animals in the institu- Facultad de Medicina Clıńica Alemana Universidad del Desarrollo. 1,2 tion. To this end, the IACUC must have current Email: [email protected] Carvajal et al. 501 and efficient access to information related to animal Materials and methods welfare provided by veterinarians and staff of the REDCap features animal facility and by the investigators responsible for the research. REDCap is a secure web application for building and The use of a paper-based system to collect data managing online surveys and databases developed at about laboratory animals in order to assess animal Vanderbilt University, and is available without cost health and welfare is a common practice among veter- for REDCap Consortium Partners. However, it is not inarians and researchers, but paper has several disad- open-source software due to agreement restrictions. vantages. To analyze changes over time in paper-based To obtain the software, a valid end-user license agree- data collection, the information must first be trans- ment between Vanderbilt University and the interested ferred to a digital spreadsheet for generating reports, institution must be reached.7 For further informa- thereby delaying decision-making in situations where tion on becoming a consortium partner, contact the animal welfare may be at risk. In addition, the separate Vanderbilt REDCap Team at the email: storage of paper records prevents information from [email protected] being easily cross-checked by the IACUC during REDCap infrastructure requires a web server with inspections, or identification of missing information PHP 5.3.0þ, a database server with MySQL 5.0þ or relevant to the IACUC mission.2 MariaDB 5.1þ or Percona Server 5.1þ, and an SMTP Advances in information technology and the wide- email server. REDCap can run on a variety of operat- spread use of the Internet have enabled the developing systems (Apache, Linux, Unix, etc.). It has no hard ment of web-based systems to manage complex requirements for server processing speed, memory, or databases related to laboratory animals.3 However, hard drive space because it requires very little initial most of these systems focus on facilitating animal pro- space (less than 10MB for both the web server and duction processes, collecting and analyzing experimen- database server combined).8 For further information tal data, and the commercial activity of animal on REDCap installation and implementation, we rec- facilities.16-19 Current systems do little to enable collab- ommend the report by Kiplin et al.9 oration with regard to the research and welfare of For login, users have their own account with access experimental animals. A web-based system aimed at only to the assigned project/database through an academic/non-profit institutions that links and facili- authentication step with a username and password. tates joint work between animal facility staff, IACUC, User activities (data entry, viewing, or changes) in and researchers for animal welfare assessment is not yet REDCap could be logged for further audit trail of available. the process.10 In 2009, Harris and colleagues reported on the devel- In order to explore the program under discussion, opment of a novel workflow methodology and software users may see and use data from an experiment using solution, aimed at rapidly developing and organizing Sprague Dawley rats at the Universidad de Desarrollo electronic data with capture tools to support clinical (Santiago, Chile) that has been recorded in the and translational research, called REDCap (Research REDCap system. Users may go into the program, Electronic Data Capture).4 Currently, REDCap is a make changes, add to it, and play with it in order to web-based system commonly used to manage data of determine how the system might benefit the user’s own clinical studies.5 However, its use in preclinical research animal research. The following is an introduction to the remains uncommon. REDCap users can collect data abovementioned animal (Sprague Dawley rats) online through any mobile device with Internet access experiment. and a web-browser (e.g. smartphone, tablet, laptop), recording data efficiently and flexibly in a structured Example of animal health monitoring format. Moreover, REDCap has multi-site access that by REDCap: Growth of female Sprague can be controlled through user profiles, ensuring Dawley rats the confidentially of information uploaded to the database.6 In order to show the properties and potentialities of the To address this issue, we used REDCap to create a REDCap application, we described an example of web-based system to collect and manage general health animal health monitoring during the breeding process data during animal breeding and experimentation, as a of female Sprague Dawley rats. The system has the key component to improve animal welfare, considering possibility to collect data of general health and the interests of the three categories of users in animal animal pain or distress indicators. However, we research: researchers, veterinarians, and members of excluded animal pain assessment since it is unnecessary the IACUC. for the system description. 502 Laboratory Animals 53(5)

Female Sprague Dawley rats were kept under con- This system is designed to be suitable for inbreeding trolled lighting from 06:00 to 18:00 hours, at a constant and/or outbreeding animal colonies. The ‘‘Breeders’’ temperature (20 to 24C), and received food (5P00 database is composed of a variable consisting of the Prolab RMH 3000, LabDiet, USA) and water ad animal’s Origin (inbreeding or purchased), Species libitum. In order to analyze animal growth, 4-week-old and Strain, Sex, Birthday, and Date and Cause of animals were weighed daily from Monday through euthanasia/death. It is possible to use the ‘‘Mating’’ Friday, every week for 7 weeks. Food and water con- database to select the breeders (male and female/s) to sumption was measured daily. Body weight, water make up the different mating types. Every litter is asso- intake, and food consumption data were collected using ciated with a specific mating (linked to the Mating data- the form entitled: ‘‘Study on the growth of female base), which enables users to track the animal pedigree. Sprague-Dawley rats at Universidad del Desarrollo’’. The ‘‘Litter&Allocation’’ database characterizes the lac- Data were captured using a local Wi-Fi signal. tation process, starting with the birthday, then weaning, and finally, allocation. At that moment, each animal is identified with a hand-made ID for tracking purposes. In Results the ‘‘Allocation’’ form, the use assigned to each animal Integrated database system design was registered under ‘‘Research,’’ ‘‘Breeder,’’ or ‘‘Euthanasia,’’ considering the ID associated with the In order to develop an appropriate database, the breed- ‘‘Litter&Allocation’’ database (Figure S1). ing process conducted by the animal facility was stu- The ‘‘Animal welfare’’ module was designed for died through staff interviews and analysis of the paper animal facility staff members, considering one room form used for data collection. In addition, paper forms as a unit inside the Animal Laboratory Facility. This used by scientists to assess animal health and/or welfare database, as mentioned before, is used to collect data during animal experimentation at the Universidad del on environmental conditions (Tmax,Tmin, and humid- Desarrollo were examined. Finally, IACUC members ity), housing management such as woodchips, food, discussed the minimal indicators needed to monitor and water change, and indicators of animal health, animal welfare during any research protocol audit. such as body weight, physical appearance, and behav- Considering the activities of the animal facility and ior. These indicators should be defined for each ani- the key indicators used to assess animal welfare, such mal’s species and strain. If any animal manifests as animal health, a database was designed with a basic symptoms of pain or discomfort, the grimace scale but flexible structure to allow changes in the selection (according to species) can be completed in the database. of welfare indicators which vary depending on the In addition, comments and photographs can automat- research study. A demonstration model of the system ically be sent to the investigator to communicate the demo is provided. See the REDCap User Guidelines in health state of the animal and help the investigator Supplementary materials. make decision about pain management or humanitar- In order to build the system’s ‘‘core,’’ the REDCap ian endpoint criteria for euthanasia (Figure 1(b)). hosted at the Universidad del Desarrollo was developed The ‘‘Animal welfare’’ form, for researchers, with three databases (Breeders, Mating, and addresses each animal individually. This form considers Litter&Allocation) to manage information related to physical and psychological indicators of animal health, animal breeding. These three databases were connected such as body weight, water and food consumption, by SQL queries in order to track animals from birth to animal appearance, and behavior. Similar to the euthanasia or death. An isolated database entitled Animal Welfare database for animal facility staff, if ‘‘Animal welfare’’ was created for use by the animal any animal manifests signs of suffering, the grimace facility staff in order to capture data related with scale can be completed, which activates the same auto- animal wellbeing such as environmental conditions matic communication mechanism with the veterinarian and animal health, including physical and nutritional to assist in joint decision-making. In addition, a ‘‘text state, clinical signs, and behavior.11 For each research box’’ was created as a space for adding comments project, a new database was generated. When required, about abnormalities in environmental conditions. animal data reported by the animal facility staff and Users will find the glossary for clinical signs and scien- researchers can be consulted and cross-referenced by tific terminology created by the FELASA Working IACUC during projects inspections (the structure of Group Report very helpful (Figure 1(a)) for creating each database is fully customizable). Each database an Institution’s monitoring protocol for experimental has a dashboard providing the status of records, animals. which provides an overview of data collection during The system can generate table reports for each user the entire activity or project. type (researcher, animal facility staff, or IACUC Carvajal et al. 503

Figure 1. Section of animal welfare indicators in (a) Animal welfare database (related to research protocol) and (b) Vivarium-Animal welfare (shown partially).

Figure 2. Schema of data flow about animal health and welfare between veterinarians, researchers, and IACUC members during a preclinical study. The direction of the blue arrows indicates who sent and receives the data or information through the REDCap system. member). For further analysis, data can be exported in Study on the growth of female the following formats: Microsoft Excel, SPSS, SAS, R, Sprague Dawley rats at the STATA, and CDISC ODM. Universidad del Desarrollo In addition, the system allows for the online submission of projects or protocols for IACUC approval. Data collected were exported to an Excel workbook, Reviewers assigned to the project can include their previously created with appropriate functions and observations/suggestions on the same platform, and pivot tables, to automatically obtain plots of body researchers can reply, revise, and submit a new version weight (%) and water and food consumption per of the research project or protocol. Finally, the IACUC animal (Supplementary Materials, Figure S2). makes the ﬁnal review and sends the research project A typing error was detected in the data collection of approval (Figure 2). body weight in animal N2 (Supplementary Materials, 504 Laboratory Animals 53(5)

Figure S2, A) and water intake in the beginning of the an efficient and collaborative manner from submission study (Supplementary Materials, Figure S2, B and C). to final approval (Table 1). In addition, a weekly periodicity in water consumption Several colony management applications based on was observed and may be explained by water leakage in relational databases have been created in recent years. cage-handling procedures. Food intake was stable The best known free applications are MouSeek,12 during the entire study. As expected, no anomalies JCMS from The Jackson Laboratory,13 and LAMA were found in physical and psychological state indica- from Vancouver University.14 They represent consider- tors in the animals assessed by the numerical score able progress in data collection, managing, and report- sheet (data not shown). Data collection was placed ing systems for animal production processes, but under the Researcher category. Research and animal function offline. Today, all commercial applications care staff monitored animal health using the abovemen- are web-based systems, such as–PyRAT Animal tioned indicators. Facility software15 and Tick@lab software,16 both Environmental conditions were measured during the used for animal research projects.17,18 These software study, and they were collected and recorded in programs mainly focus on laboratory animal facilities Supplementary Materials (Figure S3). The animal and staff, and are used to record such data as stems care staff performed data captures. In addition, a from accountability issues, task and research project research user recorded a reduction in the percentage management, animal census, and external audits. In of room humidity on 27 April (Figure 3). other words, these software programs have been designed to improve processes facilitating staff manage- Discussion ment and quality control, and are an excellent solution for the needs of industry. However, in an academic This REDCap-based system was developed to manage setting, the comprehensive, balanced, and coordinated data related to the care and use of experimental work between the abovementioned categories of users animals, and to enable the three categories of partici- (belonging to the same institution) mainly focuses on pants—researchers, animal facility staff, and ensuring animal welfare during production or experi- IACUC—to integrate and supplement information mental processes and not on facilitating commercial provided. This system promotes communication aspects of the research. between these participants about animal welfare indi- Frenzel and colleagues developed a web-based data- cators, such as environment-related risks and animal base to manage data of small animal experiments and health inside the laboratory at the animal facility and at the same time monitor animal health parameters the institution. In addition, the system facilitates animal such as animal weight and physical condition. The colony management, monitoring of animal experimen- inclusion of a warning signal in their system alerts tation, and research projects audited by the IACUC in researchers and prevents procedural errors during

Figure 3. Reports of Macro-environment data from (a) Animal facility Staff and (b) Research. Carvajal et al. 505

Table 1. Data access permissions in database system are set per each role. Icons: Data visualization, Add/Edit Data and Automatic notification (by email).

Roles Purpose of Data Databases Animal Facility Researchers IACUC

Vivarium Management - Vivarium – (Macro- & Micro-environment) Colony Management - Breeders – - Matting - Litter & Allocation Monitoring Animal Welfare - Vivarium – - Animal Welfare (Macro & Micro-environment, Animals) - Research (Animal Welfare Variables] (Related to research protocol) Research - Research – – (Testing Scientific Hypothesis) (One per each Research protocol) experiments, such as treatment overdoses, or the the protection of animals used for scientific pur- identification of animals in critical conditions that poses,21,22 and the current requirements of scientific require supportive therapies. This system is used pri- journals for better description of health monitoring marily by researchers to facilitate the collection of protocols of animals during experimentation. experimental data and manage animal welfare,19 As we mentioned before, REDCap allows user to excluding the participation of animal care staff and generate data reports. In Figure 3, we show an example IACUC members. of the comparison of reports from Research data input Carbone and Austin reviewed the descriptions of and animal care staff about environment data. As pain management after survival surgery in research ani- demonstrated, IACUC members can compare data mals in 400 scientific articles. They concluded that the quickly and easily for future studies. information provided about animal anesthetic and In summary, REDCap is a suitable tool for not- analgesic protocols is insufficient, alerting researchers for-profit institutions interested in animal production to the fact that under-appreciation of animal pain for research purposes given that it is free, adaptable could be a potential source of bias in experimental to animal laboratory facilities, and provides for the var- results, and emphasizing the necessity to treat pain.20 iety and hierarchy of research staff. Moreover, the This current system based on REDCap enables easy system enables easy communication and information generation of databases to potentially improve animal sharing between researchers, animal facility staff, and welfare, including indicators of animal health status members of the IACUC, facilitating and potentially such as body weight, food and water consumption, improving animal welfare assessment in the context of animal appearance and behavior, and pain indicators, animal experimentation. Furthermore, the system’s which vary depending on the study and the experimen- replicability and flexibility can be adapted to meet the tal species/strain.11 This system additionally collects specific requirements of institutions and research pro- and favors logical storage of data that is increasingly jects, making it an appropriate tool for analyzing data relevant at the time of publishing. not only to promote good animal wellbeing practices, The REDCap system allows the storage of warning but also to discuss and develop new recommendations messages and alerts regarding laboratory infrastructure at a global level for improving animal welfare programs failures, errors in animal proceedings, and unexpected and guidelines. animal pain or discomfort manifestations. Such information can be consulted for further consideration Acknowledgements during review of animal care and use programs or We would like to thank Isabel Abarzuá for her kind and during evaluation of the animal facility operation, its efficient support in the development of our data management environment, and other institutional factors. In add- system; Anne Bliss for her English-language assistance with ition, the system was built in accordance with the the manuscript and her great points of view of our work; Good Laboratory Practice for animal experimental Benjamin Erranz for his scientific suggestions for the manu- protocols, the European Directive (2010/63/EU) on script regarding animal breeding and welfare; and 506 Laboratory Animals 53(5)

Christopher Gumera for his accurate revision and suggestions 10. REDCap. Technical Overview. https://projectredcap.org/ for the REDCap application design. software/requirements/ (2009, accessed 17 Jan 2018). 11. Hawkins P, Morton DB, Burman O, et al. A guide to Declaration of Conflicting Interests defining and implementing protocols for the welfare The author(s) declared no potential conflicts of interest with assessment of laboratory animals: Eleventh report of respect to the research, authorship, and/or publication of this the BVAAWF/FRAME/RSPCA/UFAW Joint Working article. Group on Refinement. Lab Anim 2011; 45: 1–13. 12. Davis C. MouSeek 1.0 software. http://www.nervenet. Funding org/fmpfiles/MouSeek/MouSeekReadMe.html (2001, accessed 10 May 2017). The author(s) received no financial support for the research, 13. Donnelly CJ, McFarland M, Ames A, et al. JAX Colony authorship, and/or publication of this article. Management System (JCMS): An extensible colony and phenotype data management system. Mamm Genome ORCID iD 2010; 21: 205–215. Sebastiań D. Calligaris http://orcid.org/0000-0002-9296- 14. Milisavljevic M, Hearty T, Wong TY, et al. Laboratory 6739 Animal Management Assistant (LAMA): A LIMS for active research colonies. Mamm Genome 2010; 21: References 224–230. 15. Scionics Computer Innovation GmbH. PyRAT Animal 1. Committee for the Update of the Guide for the Care and Use of Laboratory Animals. Guide for the Care and Use Facility Software. http://www.scionics.com/pyrat.html of Laboratory Animals. 8th ed. Washington, DC, US: (2017, accessed 11 May 2017). National Research Council, The National Academic 16. a-tune Inc. TICK@LAB software for Laboratory Press, 2011, p.246. Animal Research Facilities. http://www.a-tune.com/pro- 2. Pitts M, Bayne K, Anderson L, et al. Institutional Animal ducts-services-software/ticklab-mouse-colony-manage Care and Use Committee Guidebook. 2nd ed. Office of ment-software/ (2017, accessed 11 May 2017). Laboratory Animal Welfare (OLAW) and Applied 17. Topaz Technologies. Animal Census software . http:// Research Ethics National Association (ARENA), www.topazti.com/animal-census-2 (2016, accessed 10 2002, p.230. May 2017). 3. Dagan Feng D. Biomedical Information Technology. 18. Key Solutions Company. https://http://www.keyusa. 1st ed. Elsevier, 2008, p.552. com/index.html, (2018, accessed 18 Jan 2018). 4. Harris PA, Taylor R, Thielke R, et al. Research elec- 19. Frenzel T, Grohmann C, Schumacher U, et al. tronic data capture (REDCap) – a metadata-driven Monitoring of small laboratory animal experiments by methodology and workflow process for providing trans- a designated web-based database. Lab Anim 2015; 49: lational research informatics support. J Biomed Inform 327–335. 2009; 42: 377–381. 20. Carbone L and Austin J. Pain and laboratory animals: 5. REDCap. Consortium. https://projectredcap.org/about/ Publication practices for better data reproducibility and consortium/ (accessed 8 August 2017). better animal welfare. PLoS One 2016; 11: e0155001. 6. REDCap. Software features. https://projectredcap.org/ 21. CFR. Code of Federal Regulations Title 21 – Subpart G software/ (accessed 8 May 2017). Protocol for the conduct of a nonclinical laboratory 7. REDCap. Join and get REDCap. https://projectredcap. study. http://www.accessdata.fda.gov/scripts/cdrh/cfdocs org/partners/join/ (accessed 18 Jan 2018). /cfcfr/CFRSearch.cfm?CFRPart¼58&showFR¼1&subp 8. REDCap. Requirements and dependencies for installing artNode¼21:1.0.1.1.23.7 (2016, accessed 11 May 2017). REDCap, https://projectredcap.org/software/require- 22. Directive 2010/63/EU from European Parliament and of ments/ (2006, accessed 7 May 2017). the Council on the protection of animals used for scien- 9. Klipin M, Mare I, Hazelhurst S, et al. The process of tific purposes. Official Journal of the European Union, installing REDCap, a web based database supporting 2010. biomedical research: The first year. Appl Clin Inform 2014; 5: 916–929.

Re´sume´ Les e´tudes de recherche portant sur l’expe´rimentation animale sont re´glementeés par l’Institutional Animal Care and Use Committee (IACUC). A` cette fin, l’IACUC doit inte´grer l’information fournie par les enqueˆteurs de chaque e´tude prećlinique et par les ve´te´rinaires de la structure animalie`re afin de surveiller et d’approuver le processus. Il est commun mais chronophage d’utiliser un syste`me papier pour recueillir des donneés sur la sante´ et le bien-eˆtre animal. Cette pratique est aussi sujette a` des erreurs de transcription et de lecture, et n’est pas pratique pour les ve´te´rinaires et les chercheurs qui ont besoin de prendre des dećisions collab- oratives en temps opportun lorsque le bien-eˆtre animal est menace´. Carvajal et al. 507

Nous avons creé´ un syste`me de surveillance base´ sur le web axe´ sur la sante´ animale qui a le potentiel d’ame´liorer le bien-eˆtre des animaux. Le syste`me de gestion des donneés est base´ sur le logiciel REDCap, qui permet l’inte´gration de donneés afin de proposer une solution d’e´valuation du bien-eˆtre des animaux. Le syste`me propose´ comprend les principaux indicateurs de l’e´tat de sante´ geńe´ral, comme l’environnement, les informations sur l’e´tat physique/nutritionnel, les parame`tres de comportement au cours de l’e´levage et de l’expe´rimentation, comme e´le´ments importants du bien-eˆtre des animaux. En outre, le syste`me facilite la communication de ces informations entre les chercheurs, le personnel de la structure animalie`re et l’IACUC. Le logiciel REDCap est disponible pour les organismes sans but lucratif, et peut eˆtre adapte´ et reproduit par dans les institutions inte´resseés par et responsables du bien-eˆtre des animaux, et et de leur utilisation a` des fins de recherche. Le logiciel REDCap constitue un excellent outil pour promouvoir les bonnes pratiques beńe´fiques a` la sante´ des animaux d’expe´rimentation.

Abstract Wissenschaftliche Studien mit Tierversuchen werden vom Institutional Animal Care and Use Committee (IACUC) geregelt. Zu diesem Zweck muss das IACUC die Informationen zusammenfu¨hren, die Wissenschaftler pra¨klinischer Studien und Tiera¨rzte aus Tierkliniken bereitstellen, damit der Prozess u¨ber- wacht und genehmigt werden kann. Die Verwendung eines papierbasierten Systems zur Erhebung von Daten zu Tiergesundheit und Tierschutz ist eine ga¨ngige, wenn auch zeitaufwa¨ndige Praxis mit potenziellen Transkriptions- und Lesefehlern, die zudem unpraktisch fu¨r Tiera¨rzte und Forscher ist, die zeitnahe Entscheidungen abstimmen mu¨ssen, wenn der Tierschutz auf dem Spiel steht. Wir haben ein webbasiertes Monitoringsystem mit Schwerpunkt Tiergesundheit entwickelt, das zur potenziellen Verbesserung des Tierschutzes dienen kann. Das Datenmanagementsystem basiert auf der Software REDCap, die eine Datenintegration mit Blick auf die Beurteilung des Tierschutzes ermo¨glicht. Das vorges- chlagene System umfasst Schlu¨sselindikatoren fu¨r den allgemeinen Gesundheitszustand, wie Umwelt, phy- sikalische/nahrungsbezogene Informationen und Verhaltensparameter wa¨hrend der Tierzucht und -versuche, die wichtige Komponenten des Tierschutzes sind. Daru¨ber hinaus erleichtert das System den Austausch dieser Informationen zwischen Forschern, Mitarbeitern von Tierkliniken und dem IACUC. REDCap steht gemeinnu¨tzigen Organisationen zur Verfu¨gung und kann von Institutionen mit Interesse an und Verantwortung fu¨r Tierbetreuung/-schutz, angepasst, repliziert und in der Forschung eingesetzt werden. REDCap ist ein hervorragendes Instrument zur Fo¨rderung bewa¨hrter Praktiken, die der Gesundheit von Versuchstieren dienen.

Resumen Los estudios de investigacioń que requieren la experimentacioń con animales son regulados por el Comite´ Institucional para el Cuidado y Uso de Animales de Laboratorio (CICUAL). Con este fin, el CICUAL debe integrar la informacioń suministrada por los investigadores de cada estudio preclıńico y por los veterinarios de Bioterio para poder controlar y aprobar el proceso. Utilizar un sistema en papel para recoger datos del bienestar y la salud de los animales es una praćtica comuń pero que requiere mucho tiempo, propensa a errores de lectura y transcripcioń, sin nombrar todos los inconvenientes que tiene para veterinarios e investigadores que desean tomar decisiones colaborativas y precisas cuando el bienestar del animal esta´ en peligro. Hemos creado un Sistema Web para gestioń de bases de datos que se centra en la salud animal con el potencial de mejorar su bienestar. El sistema de gestioń de datos utiliza el software REDCap, que permite la integracioń de datos para ofrecer una solucioń para la evaluacioń del bienestar animal. El programa pro- puesto incluye indicadores claves del estado de salud general, como el entorno, informaciońfı´sica/nutricional y para´metros de comportamiento durante la crıá y la experimentacioń con animales, como componentes importantes del bienestar animal. Asimismo, el sistema facilita la comunicacioń de esta informacioń entre los investigadores, el personal de Bioterio y el CICUAL. REDCap esta´ disponible para organizaciones sin fines de lucro de lucro y ada´ptandose a los requerimientos propios de las instituciones interesadas y responsables del cuidado de animales utilizados en los estudios de investigacioń. REDCap es una herramienta excelente para fomentar buenas praćticas que beneficien la salud de los animales de experimentacioń. Short Report Laboratory Animals 2019, Vol. 53(5) 508–510 ! The Author(s) 2019 Implementing strategies to reduce singly Article reuse guidelines: sagepub.com/journals- housed male mice permissions DOI: 10.1177/0023677219845028 journals.sagepub.com/home/lan Garikoitz Azkona and Juan M Caballero

Abstract There are different husbandry situations that lead to social isolation of male mice. Besides legal considerations, single housing has a negative impact on behavioural and molecular studies. We have implemented two strategies, the ‘companion mouse’ and just-weaned male grouping, to reduce the number of male mice housed singly in our facility. We have achieved a progressive reduction (42% in three years) without an increase in aggression.

Keywords individualization, male mice, companion, grouping, refinement, husbandry

Date received: 21 February 2019; accepted: 29 March 2019

Introduction Once a week socially housed mice, together with their nesting material, are transferred to clean cages.8,9 This Directive 2010/63/EU recommends the group housing same procedure is done with individually housed mice of social animals, such as mice, to maximize their well- every other week. Mice have ad libitum access to water being. However, there is an open debate about group and diet (irradiated Special Diet Services RM1 up to housing male laboratory mice.1 On one hand, it is rec- nine weeks of age, and RM3 for breeding pairs and ommended to individually house highly aggressive young mice until nine weeks old). Rooms are maintained strains of male mice to avoid fighting.2 On the other under standard environmental conditions (humidity: hand, it has been shown that single housing is not an 40–70%; temperature: 20–24C) and a 12 h light/dark optimal solution for overcoming aggression, and cycle (lights on at 08:00 h). Animals are monitored improving housing conditions could be a better every day. Animal care and use programme is approved option.3 Behavioural studies show that individually- by PRBB-Ethics Committee and accredited by housed mice display increased locomotor activity, AAALAC International, following European (2010/63/ worse habituation response and impaired memory.4 UE) and Spanish (RD 53/2013) regulations. Moreover, social isolation leads to a wide range of Females are grouped with same strain mates, but global epigenetic changes in the brain.5 adult males never are, due to aggression. Thus, when We have been improving strategies to manage indi- there is only one male with the genotype of interest in a vidually-housed mice following different recommenda- litter, it would be housed singly. To reduce the number tions.3,6 The aim of this short report is to describe of singly housed male mice we followed two strategies. the positive outcome of the protocol that we have In 2016, we started implementing the ‘companion implemented. mouse’ strategy for genetically modified mice, keeping alive a ‘companion’ littermate to avoid social isolation 10 Animals, material and methods of the single male with the genotype of interest. In 2018, we implemented a post-weaning grouping Mice born in our specific pathogen-free (SPF) breeding zone are socially housed, up to four male and five female Parc de Recerca Biome`dica de Barcelona (PRBB) Animal Facility, mice, in 1145T (Tecniplast) cages in individually venti- Spain lated cages. We use black poplar/aspen shavings as litter, Corresponding author: and irradiated tissues as nesting material. Mating pairs Juan M Caballero, Parc de Recerca Biome`dica de Barcelona – and individually-housed mice are enriched with auto- PRBB, Doctor Aiguader, 88 - 08003 Barcelona, Spain. 7 claved cardboard cylinders and cotton for nesting. Email: [email protected] Azkona and Caballero 509 strategy that consists of incorporating the single male to a litter to a cage of males with less than a week age difference.9,11 If neither of the aforementioned strategies is feasible, those mice are registered in our computer software with a welfare problem and the investigator is required to provide justification to keep them alive. Our Ethics Committee considers injuries due to fights between males, health concerns and active breeders or vasectomized males as suitable justification for individual housing. In this study, we have considered only males that were born and housed in the breeding zone to ensure that we did not include any cases of individualization due to procedural protocols. To determine statistical differences among strains we performed two-way analysis of variance followed by Bonferroni’s multiple comparisons test with Prism 6.01 (GraphPad Software). The results are described in accordance with the ARRIVE guidelines.12

Results and discussion The average number of male mice born in the breeding area of the SPF zone in the last three years was 22,299.3 938.1, corresponding to the vast majority of different lines of genetically modified mice (80.6% 2.1), followed by C57BL/6 (7.7% 0.6), NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (4.2% 0.3), SCID/Beige (3.9% 0.5) and CD1 (3.6% 0.8). Figure 1. (a) Singly housed mice (continuous line) and In 2016, we registered 2245 singly housed male mice, wounds from fights (dashed line), total number by year. In- which represented 54.4% of total welfare problems house born (b) singly housed mice and (c) wounds from fights (4128). The following year, they were 1911, 49.6% of percentage by strain. Graphs show the two periods 2016– the total welfare issues (3853), and in 2018, 1309 males, 2017, ‘companion’ strategy, and 2018, both strategies being which accounted for 40.2% of the total welfare prob- used. **p < 0.01 indicates differences between the periods. lems (3255) (Figure 1(a)). The records of singly housed NOD SCID: NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ; GM: genet- male mice three years ago were more than a half of the ically modified. total welfare problems, maybe due to the large number of in-house bred genetically altered mice. The imple- problems in 2016, 2017 and 2018, respectively mentation of the strategies led to a progressive reduc- (Figure 1(a)). There were no significant differences tion in singly housed male mice, resulting in 42% among strains (Figure 1(c)). Overall, the percentage of less individually housed male mice in 2018 compared fights related to the number of in-house born animals with 2016. represented less than 2% and in some strains close to During this period, results showed no significant dif- zero, most likely because we followed previous manage- ferences between the percentages of singly housed ani- ment recommendations.8,9 mals by strain. It is interesting to note that the implementation of the post-weaning grouping strategy Conclusion in 2018 managed to reduce the percentage to below 10% for all strains, and in some instances to below In our experience ‘companion mouse’ and just-weaned 5% (Figure 1(b)) and that this reduction is statistically male grouping strategies are suitable husbandry practices significant (F(1, 4) ¼ 26.24; p < 0.01). to reduce the number of singly housed males. In the Finally, to determine whether these measures had an future, it would be interesting to increase the age difference impact on aggression, we registered the number of to two weeks to see whether it is still possible to generate a wounds due to fights. The records indicated that the stable social group without injurious aggression or stress total number of episodes was 48, 66 and 65, corres- to the animals, as has been shown for CD19 and C57BL/ ponding to 1.2%, 1.7% and 1.9% of the total welfare 611 mice, and further reduce singly housed mice. 510 Laboratory Animals 53(5)

Acknowledgments 4. Vo˜ ikar V, Polus A, Vasar E, et al. Long-term individual The authors would like to thank Toma´s Garcia and Noray housing in C57BL/6J and DBA/2 mice: assessment of Bioinformatics, S.L.U. for welfare issues, software design and behavioral consequences. Genes Brain Behav 2005; 4: development, and Charles Rivers staff for their assistance 240–252. DOI: 10.1111/j.1601-183X.2004.00106.x. with collecting the data. 5. Siuda D, Wu Z, Chen Y, et al. Social isolation-induced epigenetic changes in midbrain of adult mice. J Physiol Pharmacol 2014; 65: 247–255. Declaration of Conflicting Interests 6. Jennings M, Batchelor GR, Brain PF, et al. Refining The author(s) declared no potential conflicts of interest with rodent husbandry: the mouse. Report of the Rodent respect to the research, authorship, and/or publication of this Refinement Working Party. Lab Anim 1998; 32: article. 233–259. DOI: 10.1258/002367798780559301. 7. Hutchinson E, Avery A and Vandewoude S. Funding Environmental enrichment for laboratory rodents. The author(s) received no financial support for the research, ILAR J 2005; 46: 148–161. authorship, and/or publication of this article. 8. Weber EM, Dallaire JA, Gaskill BN, et al. Aggression in group-housed laboratory mice: why can’t we solve the ORCID iD problem? Lab Anim (NY) 2017; 46: 157–161. DOI: 10.1038/laban.1219. Garikoitz Azkona https://orcid.org/0000-0002-5312-1207 9. Annas A, Bengtsson C and To¨rnqvist E. Group housing of male CD1 mice: reflections from toxicity studies. References Laboratory Animals 2013; 47: 127–129. 1. Kappel S, Hawkins P and Mendl MT. To Group or Not to 10. Capdevila S and Kelly H. No one likes to live alone: Group? Good Practice for Housing Male Laboratory Social housing of lab animals. ALN Mag 2016, https:// Mice. Animals (Basel) 2017; 7: 2017/11/24. DOI: recercaambanimals.wordpress.com/2016/11/12/no-one- 10.3390/ani7120088. likes-to-live-alone-social-housing-of-lab-animals/. 2. Van Loo PL, Van Zutphen LF and Baumans V. Male 11. Gaskill BN, Stottler AM, Garner JP, et al. The effect of management: Coping with aggression problems in male early life experience, environment, and genetic factors on laboratory mice. Lab Anim 2003; 37: 300–313. DOI: spontaneous home-cage aggression-related wounding in 10.1258/002367703322389870. male C57BL/6 mice. Lab Anim (NY) 2017; 46: 176–184. 3. Loo PV, Groot Ad, Zutphen LV, et al. Do male mice 12. Kilkenny C, Browne WJ, Cuthill IC, et al. Improving bio- prefer or avoid each other’s company? Journal of Applied science research reporting: The ARRIVE guidelines for Animal Welfare Science 2001; 4: 10. reporting animal research. PLoS Biol 2010; 8: e1000412.

Re´sume´ Il existe diffe´rentes situations d’e´levage qui conduisent a` l’isolation sociale de souris maˆles. Outre les conside´rations juridiques, l’isolation a un impact ne´gatif sur les e´tudes comportementales et mole´culaires. Nous avons mis en œuvre deux strate´gies, la « souris compagnon » et le regroupement de maˆles juste sevre´s, afin de re´duire le nombre de souris maˆles en cage se´pare´e dans notre e´tablissement. Nous avons obtenu une re´duction progressive (42% en 3 ans) sans augmentation de l’agression.

Abstract Unter verschiedenen Haltungsbedingungen kommt es zur sozialen Isolation von Maüsebo¨cken. Neben rechtlichen Gesichtspunkten ist zu beachten, dass Einzelhaltung negative Auswirkungen auf Verhaltens- und molekulare Studien hat. Wir haben zwei Strategien eingefu¨hrt – die ‘‘Begleitmaus’’ und Gruppenhaltung gerade entwo¨hnter Maüsebo¨cke – um die Anzahl von einzeln in unserer Einrichtung unter- gebrachten Maüsebo¨cken zu reduzieren. Wir haben eine schrittweise Reduzierung (42 % in 3 Jahren) erreicht, ohne dass versta¨rkte Auseinandersetzungen auftraten.

Resumen Durante el manejo de los ratones existen distintas situaciones que llevan al aislamiento social de los machos. Adema´s de las consideraciones legales, el mantenimiento de los ratones aislados socialmente tienen un impacto negativo, como se ha comprobado en estudios moleculares y de comportamiento. En nuestro animalario hemos implementado dos estrategias: el denominado ‘‘ratońacompanãnte" y agrupar los machos recień destetados, a fin de reducir el nu´mero de ratones individualizados. Hemos conseguido una reducciœn progresiva (42% en 3 anõs) sin un aumento de las agresiones. Expert in the world of research diets

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News Laboratory Animals 2019, Vol. 53(5) 513 ! The Author(s) 2019 Article reuse guidelines: Watch out for three FELASA Working sagepub.com/journals- permissions Group recommendations DOI: 10.1177/0023677219872667 journals.sagepub.com/home/lan

Jean-Philippe Mocho

‘Revised recommendations for health monitoring of non-human primate colonies (2018): FELASA Working Group Report’ by Ivan Balansard (convener), Lorna Cleverley, Keith Cutler, Mats Spa˚ngberg, Kevin Thibault-Duprey and Jan Langermans. The rodent genetics Working Group finalized their extensive recommendations with ‘Genetic quality assurance and genetic monitoring of laboratory mice and FELASA Working Groups are very active under the rats. FELASA Working Group Report’ by Fernando supervision of Vice-President Working Groups Benavides (convener), Thomas Ru¨licke, Jan-Bas Prins, Martina Dorsch. There are currently about a dozen James Bussell, Ferdinando Scavizzi, Paolo Cinelli, active Working Groups listed on the dedicated web- Yann Herault and Dirk Wedekind. page: http://www.felasa.eu/working-groups. Topics for The hard work of the joint FELASA/COST action future Working Groups are discussed in every with EuFishBioMed on zebrafish husbandry resulted in FELASA Board of Management meeting, Terms of ‘Zebrafish: Housing and husbandry recommendations’ Reference are then drafted and Working Group mem- by Peter Alestro¨m, Livia D’Angelo, Paul Midtlyng, bers are selected following their nominations by Daniel Schorderet, Stefan Schulte-Merker, Frederic FELASA member associations. Sohm and Susan Warner. Most Working Groups have two years to write some The Working Group on ‘Farm animals – Health and recommendations, which are published in Laboratory welfare of ruminants and pigs’ with Corina Mihaela Animals. Lately, FELASA started to sponsor these rec- Berset (Convenor), Maria Emiliana Caristo, Fabienne ommendations’ Open Access under the Creative Ferrara, Patrick Hardy, Marianne Oropeza-Moe and Commons Attribution 4.0 License (CC BY 4.0). This Ryan Waters is going to finalize its report soon. allows anyone to read, share and re-use the FELASA All these FELASA Working Groups’ recommenda- recommendations at no cost without permission as long tions are published in Laboratory Animals and are as the publication is properly referenced. This year, available on-line ahead of the printed journal. Have a three Working Groups’ recommendations have been good read! approved. This licence allows others to re-use the Contribution. Jean-Philippe Mocho The Working Group on health monitoring of non- FELASA Honorary Secretary, FELASA, UK human primates concluded their good work with the

FELASA Honorary Secretary, FELASA, Eye, UK

Corresponding author: J-P Mocho, PO Box 372, Eye, IP22 9BR, UK. Email: [email protected]

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Meetings of interest to laboratory animals scientists and technicians: references to Laboratory Animals are for further details. Items for inclusion should be sent to Notes and Comments Editor, LAL, PO Box 373, Eye, Suffolk, IP22 9BS, UK. Email to [email protected]. The deadlines for inclusion of material are: February issue, 10 November; April issue, 10 January; June issue, 10 March; August issue, 10 May; October issue, 10 July; December issue, 10 September. 2019

1 October Zebrafish Husbandry Association UK meeting, London, UK. For further information visit https://www.nc3rs.org.uk/ events/zebrafish-husbandry-association-uk-meeting 2 October NC3Rs/IAT Animal Technicians’ Symposium 2019, London, UK. For further information visit https://www.nc3rs. org.uk/events/nc3rsiat-animal-technicians-symposium-2019 2–4 October 45th AFSTAL Conference, La Rochelle, France. For further information visit https://www.colloque-afstal.com/2019/ 4 October Severity and humane endpoints in fish research, Bergen, Norway. For further information visit https://www. fondazioneguidobernardini.org/en/programs/course-severity-and-humane-endpoints-in-fish-research.html 8–10 October Laboratory Animal Anaesthesia and Perioperative Care Workshop, Newcastle-upon-Tyne, UK. For further information see https://forms.ncl.ac.uk/view.php?id¼4790 10–11 October 3rd RSPCA international meeting: Focus on severe suffering - Avoiding mortality, Athens, Greece. For further information visit https://science.rspca.org.uk/documents/1494935/9042554/Advertþ-þFoSSþAthensþ2019.pdf/ 7e76bb5e-d5bb-885f-caed-199c77598a3b?t¼1559554582826 10–13 October 22nd European Congress on Alternatives to Animal Testing / 19th Annual Congress of EUSAAT, Linz, Austria. For further information visit http://eusaat-congress.eu/ 13–17 October AALAS National meeting, Denver, Colorado, USA. For further information visit https://www.aalas. org/nationalmeeting/general-information/future-meetings 21 October The Applicability of In Silico Models to Support In Vitro Studies, Leeds, UK. For further information see http://www. ivts.org.uk/site/ivts-workshop/ 29 October European Partnership for Alternative Approaches to Animal Testing (EPAA) 2019 Annual Conference, Brussels, Belgium. For further information see https://ec.europa.eu/growth/sectors/chemicals/epaa_en 5 November RSPCA/UFAW Rodent and Rabbit Welfare Meeting, Birmingham, UK. For further information contact research. [email protected] 6–8 November SECAL National Congress. Sevilla. Spain. For further information visit https://www.secalsevilla2019.com 8 November 2019 Primate Welfare Meeting, London, UK. For further information see https://www.nc3rs.org.uk/events/2019- primate-welfare-meeting 17–21 November American College of Toxicology Annual Meeting (ACT 2019), Phoenix, Arizona, USA. For further information visit https://www.actox.org/am/am2019/index.asp 19 November UK Biobanking Showcase, Nottingham, UK. For further information see https://biobankinguk.org/uk-biobanking- showcase-2019/ 21–22 November Organising and operating activities in a laboratory animal facility: Critical points and bottlenecks, Milan Italy. For further information visit https://www.fondazioneguidobernardini.org/en/programs/course-organizing-and-operating-activities-in-a-aboratory-animal-facility-ii-critical-points-and bottlenecks.html 26–28 November LASA Annual meeting, Birmingham, UK. For further information visit http://www.lasa.co.uk/meetings/ 2 December Organ-on-a-Chip: Current Gaps and Future Directions, Stevenage, UK. For further information visit https://www. eventsforce.net/biochemsoc/frontend/reg/thome.csp?pageID¼28848&eventID¼65&traceRedir¼2 13–14 December National Conference on Alternatives to Animal Experiments, Mumbai, India. For further information visit http:// www.saae-i.org/events.php

2020

23–26 March Animal Science and Technology (AST) Conference 2020, Edinburgh, UK. For further information visit https://www. ast2020.org/ 22-23 June LASA animal science (transgenics) section workshop: cryopreservation and IVF technology. Venue TBA. For further information contact [email protected] 23–27 August 11th World Congress on Alternatives and Animal Use in the Life Sciences, Maastricht, The Netherlands. For further information see http://wc11maastricht.org/ 25–29 October AAALAS National meeting, Charlotte, NC. Further information to follow.

Index to Advertisers OCTOBER 2019

AALAS 420 Fine Science Tools GmbH 416 SAFE/J Rettenmaier & So¨hne 427 Altromin International OBC SECAL 517 AnLab Ltd 512 IPS Product Supplies Ltd 422 SGV 519 AST2020 522, 523 Laboratory Animals Ltd (LAL) 414, 521 ssniff Spezialdia¨ten GmbH 425 Avid (Labtrac) 495 Laboratory of Pharmacology and Toxicology Special Diets Services 511 Bell Isolation Systems Ltd 516 GmbH & Co KG 419 Tecniplast SpA IBC LBS 426 Transnetyx Inc 415 CEDS 512 Charles River Laboratories IFC Marshall BioResources 428 Vet-Tech Solutions Ltd 518 Matachana Germany GmbH 421 Datesand Ltd 424 ZOONLAB 423 PFI Systems Ltd 520 ECLAM 515

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