Biological Containment for African Swine Fever (ASF) Laboratories and Animal Facilities: The Italian Challenge in Bridging the Present Regulatory Gap and Enhancing Biosafety and Biosecurity Measures.

The African Swine Fever Virus (ASFV) is a DNA virus of the Asfarviridae family, Asfivirus genus. It is responsible for massive losses in pig populations and drastic direct and indirect economic impacts. The ever-growing handling of ASFV pathological material in laboratories, necessary for either diagnostic or research activities, requires particular attention to avoid accidental virus release from laboratories and its detrimental economic and environmental effects. Recently, the Commission Delegated Regulation (EU) 2020/689 of 17 December 2019 repealed the Commission Decision of 26 May 2003 reporting an ASF diagnostic manual (2003/422/EC) with the minimum and supplementary requirements for ASF laboratories. This decision generated a regulatory gap that has not been addressed yet. This paper aims to describe the Italian National Reference Laboratory (NRL) efforts to develop an effective and reliable biological containment tool for ASF laboratories and animal facilities. The tool consists of comprehensive and harmonized structural and procedural requirements for ASF laboratories and animal facilities that have been developed based on both current and repealed legislation, further entailing a risk assessment and internal audit as indispensable tools to design, adjust, and improve biological containment measures.

Perceived professional quality of life and mental well-being among animal facility personnel in Spain.

Animal facility personnel provide the husbandry and care of laboratory animals. We aimed to investigate their work-related quality of life, empathy and mental well-being. Participants living in Spain were contacted by email and asked to complete an anonymous online questionnaire, in which they answered the Professional Quality of Life scale, the Cognitive and affective empathy test, the Warwick-Edinburgh Mental Well-Being Scale, and their perceived human-animal interaction. Participants were asked whether they were receiving psychological therapy or were taking anxiolytics, hypnotics or antidepressant medication. The study comprised 80 participants. No differences were observed related to personal or professional variables. Participants working with small carnivores reported higher total empathy, and those working with non-human primates reported higher emotional comprehension. Higher human-animal interaction was reported by participants working with small carnivores, farm animals and non-human primates. More than half of the participants reported high levels of mental well-being, positively correlated with emotional comprehension, emphatic joy and compassion satisfaction. Participants working with farm animals reported higher levels of secondary traumatic stress that was positively correlated with human-animal interaction and negatively with mental well-being. Most participants reported low-average levels of burnout, which was negatively correlated with mental well-being. The percentage of animal facility personnel in psychotherapy was higher than in the general population, and the consumption of anxiolytics was a little lower and antidepressants higher. Overall, our results indicate that animal-facility personnel who felt stress or worse mental well-being were in therapy and took medication to improve their condition.

A survey of the impact of COVID-19 on the management of animal experiments and laboratory animal facilities in Korea.

The pandemic has affected the lives of people all over the world. The effects of the pandemic on laboratory animal facilities and their operations through this unusual global event are poorly understood. Here, we have applied a methodological framework of qualitative approach including semi-structured interviews to investigate laboratory animal operations in Korea and how it has shaped the on-going management and laboratory operations of such facilities. A total of fifty-two individuals, including members and administrators of the Institutional Animal Care and Use Committee (IACUC), researchers, and animal facility managers and staff, were surveyed through purposeful sampling. Survey questions explored how the pandemic impacted the IACUC and the functioning of animal facilities, and what steps to take in preparation of a future pandemic-like crisis. Our survey found evidence of an increase in animal experiments in Korea during the pandemic that correlated with increases in research funding during that period, such as for vaccine development. Also, operational challenges due to pandemic-related health issues in personnel were resolved through overtime, rather than by reducing facility operations. Moreover, a refinement of post-approval monitoring (PAM) practices was also discussed by respondents. Taken together, our study offers insights into animal facility operations during the pandemic and outlines recommendations for safeguarding operations in such future scenarios.

An Outbreak of the Nematode Parasite Eustrongylides spp. (Nematoda: Dioctophymatidae) in a Zebrafish (Danio rerio) Facility.

Despite advances in research employing zebrafish (Danio rerio) as an animal model, it is still difficult to obtain animals with attested sanitary qualities from commercial breeders. This study reports the first-time occurrence of Eustrongylides spp. parasitism in a zebrafish colony recently acquired from a pet store supplier to begin a research colony at a scientific facility. This parasite has not been reported in any current zebrafish health-monitoring guidelines to date. Thus, this report is a warning to breeders and researchers that this nematode can parasitize zebrafish, causing high lethality rates and compromising research outcomes.

Mouse breeding facilities in Argentina: Current state, challenges, and strengths in relation to animal welfare.

The science and technology of laboratory animals has come a long way worldwide, but for reasons related to the development of the countries, this journey started later in some Latin American countries, as is the case of Argentina. Without a specific legal framework to conduct animal experimentation, local strengths to promote animal welfare are based on professionals specifically trained in the care of laboratory animals as well as an extended network of ethics committees that ensures compliance with the ethical principles applied to animal experimentation. Nevertheless, there are no updated reports showing welfare indicators in rodent facilities. Therefore, we conducted a survey on mice breeding facilities enrolled in a national record elaborated by the National Ministry of Science. Questions related to four of the Five Domains Model of Mellor, concerning (1) nutrition, (2) physical environment, (3) health, and (4) behavioral interactions with the environment, other animals, and humans, were included as well as information concerning general aspects of the establishments. Data obtained from 25 mice breeder facilities localized all over the country were summarized, providing for the first time a clear picture of the national situation about the welfare of laboratory mice in these establishments. This data will be essential to design future policy as well as for deciding priorities aiming to improve the welfare of mice bred in Argentinian facilities.

El "barbering": un problema frecuente en ratones de laboratorio / Barbering: a frequent problem in laboratory mice / O comportamento de barbeiro: um problema frequente in camundongos de laboratório

Resumen El barbering es una conducta que consiste en halar o recortar el pelo y en algunos casos incluso ingerirlo. Este comportamiento ha sido reportado tanto en el ser humano como en otras especies, no obstante, su presentación en ratones de laboratorio es frecuente en algunos linajes, genera impacto sobre su bienestar y la fiabilidad de los resultados de investigación, siendo un problema destacado en los bioterios de ratones. Al realizar una revisión de literatura disponible sobre esta condición, se identifican múltiples causas, entre estas, deficiencias en el enriquecimiento ambiental, alteraciones de manejo, estrés, conductas aprendidas, ansiedad y dominancia, así como causas genéticas haciendo que algunas cepas, linajes y modelos genéticamente modificados sean más susceptibles al barbering y ayudando a facilitar la comprensión de rutas metabólicas involucradas en esta condición. La etiología múltiple del barbering permite abordar opciones de manejo orientadas hacia la intervención ambiental y algunas estrategias farmacológicas que podrían ser útiles para reducir su incidencia y los problemas de salud y desempeño reproductivo asociados.

Abstract Barbering is a behavior that consists of pulling, cutting, or even ingesting hair. This behavior has been reported in humans or other animals; nevertheless, its presentation in laboratory mice is common in some mice strains, affects these animals' welfare, and results of research studies being a problem in mice facilities. After reviewing the available literature on this condition, multiple causes are identified, including deficiencies in environmental enrichment, management alterations, stress, learned behaviors, anxiety, and dominance, as well as genetic causes, making some strains, lineages, and genetically modified models more susceptible to barbering and helping to facilitate the understanding of metabolic pathways involved in this condition. The multiple etiology of barbering makes it possible to approach management options oriented towards environmental intervention and some pharmacological strategies that could be useful to reduce its incidence and associated reproductive health and performance problems.

Resumo O comportamento de barbeiro consiste em puxar ou aparar o pelo e, em alguns casos, até mesmo ingeri-lo. Esse comportamento tem sido relatado tanto em humanos quanto em outras espécies, entretanto, sua apresentação em camundongos de laboratório é frequente em algumas linhagens, gerando impacto no seu bem-estar e na confiabilidade dos resultados das pesquisas, sendo um problema de destaque nos biotérios. Ao realizar uma revisão da literatura disponível sobre esta condição, múltiplas causas foram identificadas, incluindo deficiências no enriquecimento ambiental, alterações de manejo, estresse, comportamentos aprendidos, ansiedade e dominância, além de causas genéticas, tornando mais suscetíveis à barbearia à algumas linhagens e modelos geneticamente modificados e ajudando a facilitar o entendimento das vias metabólicas envolvidas nessa condição. A múltipla etiologia deste comportamento de barbeiro permite abordar opções de gestão orientadas para a intervenção ambiental e algumas estratégias farmacológicas que podem ser úteis para reduzir a sua incidência e os problemas de saúde e desempenho reprodutivos associados.

Sclerocarya birrea caffra nut meal as a substitute to soyabean meal: Effects on growth performance, feed intake and utilisation and viscera macromorphometry of blue-helmeted guinea fowl.

Imported soyabean meal (SBM) is the major dietary protein (DP) source for the sub-Saharan African poultry industry making poultry production costly. Therefore, alternative locally available DP sources are required. We evaluated the potential of locally available Marula nut meal (MNM) to substitute SBM in Guinea fowl (GF) diets by determining its effects on growth, feed intake (FI) and utilisation and viscera macromorphometry. Five grower diets wherein, on a CP basis, MNM substituted SBM at 0, 25, 50, 75 and 100% were formulated. Thirty-eight 4-week-old keets (n = 7 - 8), each individually housed in a cage, were randomly assigned to grower diets, and fed for 5 weeks and then transferred to corresponding finisher diets and fed for 3 weeks. Induction and weekly body mass, daily FI, and terminal body mass (TBM), body mass gain (BMG), average daily gain (ADG) and feed conversion ratio (FCR) were determined. On slaughter, viscera masses, small and large intestines lengths, tibiae and femora indices were determined. In week 2 of the grower phase GF fed diet 3 (50% MNM CP) had the highest weekly BMG and ADG (P < 0.05) and in week 5 GF fed diet 5 (100% MNM CP) had the highest FI (P < 0.05). Dietary MNM did not affect the GF's BMG, ADG, FI and FCR during weeks 1, 3 and 4 of the grower phase. In week 3 of the finisher phase GF fed diet 3 (50% MNM CP) had the highest (P < 0.05) FCR. Dietary MNM had no effect (P > 0.05) on the trial BMG, ADG and FI of the GF but GF reared on grower and finisher diets 3 (50% substitution of SBM CP) had the highest (P < 0.05) FCR. MNM had no effect on tibiae and femora masses, lengths, and mass: length ratios and viscera macromorphometry of the GF. We conclude that MNM can, on a CP basis, substitute SBM, in GF grower and finisher diets at 25%, 75% and 100% without compromising growth, FI and utilization and viscera of GF.

COVID-19 disaster management plans for two laboratory animal facilities in South Africa.

Policies and guidelines are available for acute disasters such as earthquakes, fire and floods, however, little is available on how laboratory animal facilities should mitigate subacute disasters like the COVID-19 pandemic that imposed major restrictions on the free movement of people. As such, laboratory animal facilities had to find plausible mitigating measures to safeguard the welfare of animals in their care, to prevent animal suffering if staff could not reach the animals, albeit with limited time. The simplest approach was to stop active experiments and halt animal breeding, or to euthanize all animals. Challenges with such methods included the ethical debate regarding euthanasia of animals at the start of a pandemic and the need to perform a harm-benefit analysis while drafting the disaster plans, termination of studies at advanced stages with information loss or killing of genetically modified strains that would be difficult to replace.Two research animal facilities in South Africa addressed these challenges by implementing several changes such as allowing only essential studies to continue, maintaining small breeding colonies for essential strains, and providing staff with private transport for travelling to and from work to avoid public transport and risk of exposure to SARS-CoV-2. Engineering changes included redesigning working areas to cater for social distancing.The mitigating measures put in place by the two laboratory animal facilities were successful in ensuring the continued welfare of animals during the COVID-19 lockdown restrictions. These measures can be adopted in future pandemics that lead to restricted movement of staff.Plans de gestion en cas de catastrophes telles que le COVID-19 pour deux installations d'animaux de laboratoire en Afrique du Sud.

Vaporized Hydrogen Peroxide Decontamination of N95 Respirators in a Dedicated Animal Research Facility for Reuse During a Novel Coronavirus Pandemic.

Introduction: During the COVID-19 pandemic, health care systems and safety providers have faced an unprecedented challenge of limited access to personal protective equipment (PPE) to conduct patient and public care. In federal emergencies, reuse of PPE after disinfection can occur by processes, like vaporized hydrogen peroxide (VHP), recommended by the Centers for Disease and Control and Prevention. We identified a vacant animal holding facility at our institution to repurpose into a regional VHP decontamination center. Methods: The facility is a multiroom, 20 000 ft2 building with control of HVAC to adjust to VHP conditional requirements. H2O2 was delivered to rooms using robotic HaloFoggers, dispersing H2O2 vapor and increasingly concentrated microdroplets as a fog for a timed period based on cubic footage of rooms. Results: Fogging cycles eliminated 6-log Geobacillus stearothermophilus up to 7 days postcycle. Functional efficacy of treated N95s was confirmed by fit tests of institutional personnel. Signage, process flow mapping, and training materials facilitated ease of workflow and adherence to safety expectations within the building. Discussion and Conclusion: Our study determined that a variety of N95 respirator types and sizes were able to be cleared of potential bacterial and viral agents using VHP in a controlled fog/dwell/exhaust cycle. This repurposed animal facility has the capacity to decontaminate up to 6700 respirators daily, which will address the predicted surge of COVID-19 cases in the state, and ultimately allow each respirator to be reused multiple times. There is no other public site in the region with our capacity to offset the continued supply chain issues for PPE needs.

Assessment of environmental and occupational exposure while working with multidrug resistant (MDR) fungus Candida auris in an animal facility.

In less than a decade since its identification in 2009, the emerging fungal pathogen Candida auris has become a major public health threat due to its multidrug resistant (MDR) phenotype, high transmissibility, and high mortality. Unlike other Candida species, C. auris has acquired high levels of resistance to an already limited arsenal of antifungals. As an emerging pathogen, there are currently a limited number of documented murine models of C. auris infection. These animal models use inoculums as high as 107-108 cells per mouse, and the environmental and occupational exposure of working with these models has not been clearly defined. Using real-time quantitative polymerase chain reaction (PCR) and culture, we monitored the animal holding room as well as the procedure room for up to 6 months while working with an intravenous model of C. auris infection. This study determined that shedding of the organism is dose-dependent, as detectable levels of C. auris were detected in the cage bedding when mice were infected with 107 and 108 cells, but not with doses of 105 and 106 cells. Autoclaving bedding in closed micro-isolator cages was found to be an effective way to minimize exposure for animal caretakers. We found that tissue necropsies of infected mice were also an important source of potential source exposure to C. auris. To mitigate these potential exposures, we implemented a rigorous "buddy system" workflow and a disinfection protocol that uses 10% bleach followed by 70% ethanol and can be used in any animal facility when using small animal models of C. auris infection.

Pathogen elimination and prevention within a regulated, Designated Pathogen Free, closed pig herd for long-term breeding and production of xenotransplantation materials.

BACKGROUND: We established a Source Animal (barrier) Facility (SAF) for generating designated pathogen-free (DPF) pigs to serve as donors of viable organs, tissues, or cells for xenotransplantation into clinical patients. This facility was populated with caesarian derived, colostrum deprived (CDCD) piglets, from sows of conventional-specific (or specified) pathogen-free (SPF) health status in six cohorts over a 10-month period. In all cases, CDCD piglets fulfilled DPF status including negativity for porcine circovirus (PCV), a particularly environmentally robust and difficult to inactivate virus which at the time of SAF population was epidemic in the US commercial swine production industry. Two outbreaks of PCV infection were subsequently detected during sentinel testing. The first occurred several weeks after PCV-negative animals were moved under quarantine from the nursery into an animal holding room. The apparent origin of PCV was newly installed stainless steel penning, which was not sufficiently degreased thereby protecting viral particles from disinfection. The second outbreak was apparently transmitted via employee activities in the Caesarian-section suite adjacent to the barrier facility. In both cases, PCV was contained in the animal holding room where it was diagnosed making a complete facility depopulation-repopulation unnecessary. METHOD: Infectious PCV was eliminated during both outbreaks by the following: euthanizing infected animals, disposing of all removable items from the affected animal holding room, extensive cleaning with detergents and degreasing agents, sterilization of equipment and rooms with chlorine dioxide, vaporized hydrogen peroxide, and potassium peroxymonosulfate, and for the second outbreak also glutaraldehyde/quaternary ammonium. Impact on other barrier animals throughout the process was monitored by frequent PCV diagnostic testing. RESULT: After close monitoring for 6 months indicating PCV absence from all rooms and animals, herd animals were removed from quarantine status. CONCLUSION: Ten years after PCV clearance following the second outbreak, due to strict adherence to biosecurity protocols and based on ongoing sentinel diagnostic monitoring (currently monthly), the herd remains DPF including PCV negative.

Disaster Planning for Animals in Hazardous Agent Containment Units.

Disaster response planning for laboratory animal facilities is a time- and personnel-intensive undertaking. This article outlines numerous considerations in formulating a plan for disaster response in a high containment animal unit. The planning process is discussed around a set of elements: planning team formation, situational understanding, goal and objective determination, plan development, preparation, and rehearsal or implementation. The importance of an appropriate planning team and personnel development is explored in relationship to exemplary disaster scenarios such as natural disaster and terrorism. Specific risks such as hazardous agent and animal species type serve to delineate goal-setting methods. These goals provide the framework for an institutional disaster plan. The review further uses elements of the planning process to explore the difficulties of euthanasia of animals treated with hazardous agents. Ultimately, the pitfalls of handling media relations following disaster are examined. Proactive measures for preparing to speak to the media and mitigate negative perceptions of research are presented.

Spontaneous listeriosis in grey mouse lemurs (Microcebus murinus), but not in Goodman's mouse lemurs (Microcebus lehilahytsara) of the same colony.

Listeriosis is a zoonotic infection with the gram positive, facultative intracellular bacterium Listeria (L.) monocytogenes. Infections mainly occur in ruminants, but also in other species, including humans. Case fatality rate usually is high. The incidence of listeriosis in captive non-human primates is very low. We report the first spontaneous, fatal, and likely food-born outbreak of listeriosis in a population of captive grey mouse lemurs (Microcebus murinus). Conspicuously, none of the closely related Goodman's mouse lemurs (Microcebus lehilahytsara) in the same facility were affected.

The biomechanical overload of the upper limb: a neglected occupational hazard in animal facility operators.

Data on biomechanical overload of the upper limb in animal facility operators are currently scanty. We decided to study this risk in a university animal facility. Eleven different tasks performed by operators were identified. For each of them, the biomechanical overload of the upper limb was evaluated by applying 4 different methods frequently used, hypothesising a task duration of 4 and 8 h. Then two 'typical' real working days of the examined facility were reconstructed, and the risk for operators was calculated using the OCRA Index, Checklist and Mini-Checklist. Considering the specific tasks, the results show some difference among methods, but the overall results show an acceptable/slight risk of biomechanical overload of the upper limb in animal facility operators during typical working days. Practitioner Summary: Upper limb biomechanical overload (UL-BO) is a neglected risk in animal facilities. In a university facility, 11 different tasks were identified, and 2 typical working days were analysed. Even if some task at increased risk may exist, during typical working days,  the overall results show that the risk of UL-BO in operators can be considered usually acceptable or, at worst, slight.

Heterogeneity of the gut microbiome in mice: guidelines for optimizing experimental design.

Targeted manipulation of the gut flora is increasingly being recognized as a means to improve human health. Yet, the temporal dynamics and intra- and interindividual heterogeneity of the microbiome represent experimental limitations, especially in human cross-sectional studies. Therefore, rodent models represent an invaluable tool to study the host-microbiota interface. Progress in technical and computational tools to investigate the composition and function of the microbiome has opened a new era of research and we gradually begin to understand the parameters that influence variation of host-associated microbial communities. To isolate true effects from confounding factors, it is essential to include such parameters in model intervention studies. Also, explicit journal instructions to include essential information on animal experiments are mandatory. The purpose of this review is to summarize the factors that influence microbiota composition in mice and to provide guidelines to improve the reproducibility of animal experiments.

Sex-dependent effects of maternal separation on plasma corticosterone and brain monoamines in response to chronic ethanol administration.

Prolonged and repeated periods of maternal separation produce behavioral phenotype of increased vulnerability to neuropsychiatric disorders and drug abuse. Most of the changes in behavior, corticosterone (CORT) and monoamine levels induced by long maternal separation (LMS) are observed after a challenge, but not in basal conditions. LMS increases ethanol-induced locomotor response and self-administration, possibly due to changes in CORT release and/or monoamine concentrations. This study examined the effects of LMS in association with chronic ethanol treatment on plasma CORT and brain monoamine concentrations in male and female Swiss mice, which were kept undisturbed (animal facility rearing - AFR) or separated from their mothers for 3h/day, from 2 to 14 days of age (LMS). As adults, one set of male and female mice received no drug treatment to assess the effect of LMS per se. Another set of animals received saline injections for 20 days and one ethanol injection (2.2g/kg, i.p.) on day 21 (acute) or ethanol for 21 days (chronic). Locomotor activity, plasma CORT levels and monoamines in the frontal cortex, striatum and hippocampus of AFR and LMS mice were evaluated in non-treated, acute and chronic ethanol-treated animals. In non-treated mice, no differences were found in CORT or locomotor activity, with small changes in monoamines content. In LMS females, chronic ethanol increased dopamine and serotonin concentrations in the frontal cortex, relative to acute ethanol LMS and to chronic ethanol-treated AFR groups (p<0.05). In LMS males, chronic ethanol increased hippocampal noradrenaline, dopamine, serotonin and metabolites when compared to respective AFR controls, as well as acute LMS. Moreover, chronic ethanol treatment resulted in higher CORT concentrations in LMS than in AFR males. Overall, these results indicate that LMS mice were more susceptible to the effects of chronic ethanol administration on CORT and brain monoamine concentrations, and that these effects were sex-dependent.

Postpartum Behavioral Profiles in Wistar Rats Following Maternal Separation - Altered Exploration and Risk-Assessment Behavior in MS15 Dams.

The rodent maternal separation (MS) model is frequently used to investigate the impact of early environmental factors on adult neurobiology and behavior. The majority of MS studies assess effects in the offspring and few address the consequences of repeated pup removal in the dam. Such studies are of interest since alterations detected in offspring subjected to MS may, at least in part, be mediated by variations in maternal behavior and the amount of maternal care provided by the dam. The aim of this study was to investigate how daily short (15 min; MS15) and prolonged (360 min; MS360) periods of MS affects the dam by examining postpartum behavioral profiles using the multivariate concentric square field (MCSF) test. The dams were tested on postpartum days 24-25, i.e., just after the end of the separation period and weaning. The results reveal a lower exploratory drive and lower risk-assessment behavior in MS15 dams relative to MS360 or animal facility reared dams. The present results contrast some of the previously reported findings and provide new information about early post-weaning behavioral characteristics in a multivariate setting. Plausible explanations for the results are provided including a discussion how the present results fit into the maternal mediation hypothesis.