FELASA-AALAS Recommendations for Biosecurity in an Aquatic Facility, Including Prevention of Zoonosis, Introduction of New Fish Colonies, and Quarantine.

FELASA and AALAS established a joint working group to advise on good practices for the exchange of fish for research. In a first manuscript, the working group made recommendations for health monitoring and reporting of monitoring results. The focus of this second related manuscript is biosecurity in fish facilities. First, we define the risk of contamination of personnel by zoonotic pathogens from fish or from system water, including human mycobacteriosis. Preventive measures are recommended, such as wearing task-specific personal protective equipment. Then we discuss biosecurity, highlighting the establishment of biosecurity barriers to preserve the health status of a facility. A functional biosecurity program relies on integration of the entire animal facility organization, including the flow of staff and animals, water treatments, and equipment sanitation. Finally, we propose 4 steps for introducing new fish colonies: consideration of international trade and national restrictions; assessing risk according to fish source and developmental stage; establishing quarantine barriers; and the triage, screening, and treatment of newly imported fish. We then provide 3 realistic sample scenarios to illustrate practical biosecurity risk assessments and mitigation measures based on considerations of health status and quarantine conditions.

FELASA-AALAS Recommendations for Monitoring and Reporting of Laboratory Fish Diseases and Health Status, with an Emphasis on Zebrafish (Danio Rerio).

The exchange of fish for research may expose an aquatic laboratory to pathogen contamination as incoming fish can introduce bacteria, fungi, parasites, and viruses capable of affecting both experimental results and fish and personnel health and welfare. To develop risk mitigation strategies, FELASA and AALAS established a joint working group to recommend good practices for health monitoring of laboratory fish. The recommendations address all fish species used for research, with a particular focus on zebrafish (Danio rerio). First, the background of the working group and key definitions are provided. Next, fish diseases of high impact are described. Third, recommendations are made for health monitoring of laboratory fishes. The recommendations emphasize the importance of daily observation of the fish and strategies to determine fish colony health status. Finally, report templates are proposed for historical screening data and aquatic facility description to facilitate biohazard risk assessment when exchanging fish.

HLD-NAC design and evaluation of a fully dilutable lecithin-linker SMEDDS for ibuprofen.

Lecithin-linker microemulsions have been previously proposed as a platform for designing a fully dilutable self-microemulsifying drug delivery system (SMEDDS). This SMEDDS formulation, composed of ethyl caprate (oil), lecithin (Le), glycerol monooleate (lipophilic linker, LL) and polyglycerol caprylate (hydrophilic linker, HL), produced a ternary phase diagram (TPD) that had a fully dilutable path suitable for oral drug delivery. However, introducing ibuprofen as an active pharmaceutical ingredient (API) resulted in TPD phase boundaries that eliminated the fully dilutable path. The purpose of this work was to understand the origin of the changes in the TPD, use that understanding to restore the fully dilutable path with an ibuprofen-loaded SMEDDS, and finally to evaluate the absorption of ibuprofen in vivo. The effect of ibuprofen on the HLD (hydrophilic-lipophilic difference, interpreted as normalized net interfacial curvature) of the system was evaluated via a polar oil model, showing that ibuprofen played a surfactant-like role, having a characteristic curvature (Cc) value of +5 (highly hydrophobic). The net-average curvature (NAC) framework used the HLD calculated with Le, LL, HL and ibuprofen Cc to generate TPDs in ibuprofen lecithin-linker systems. The HLD-NAC simulations show that restoring full dilutability required a highly hydrophilic linker (HL-) with a Cc of -5 or more negative. The fully dilutable path was restored after introducing a hexaglycerol caprylate as HL- (Cc = -6). Plasma concentration profiles obtained with this ibuprofen-loaded SMEDDS showed a more than three-fold increase in the area under the curve (AUC) of rat plasma concentration profiles compared to the same 25 mg/kg ibuprofen dose in suspension.

Cage-lid hanging behavior as a translationally relevant measure of pain in mice.

ABSTRACT: The development of new analgesic drugs has been hampered by the inability to translate preclinical findings to humans. This failure is due in part to the weak connection between commonly used pain outcome measures in rodents and the clinical symptoms of chronic pain. Most rodent studies rely on the use of experimenter-evoked measures of pain and assess behavior under ethologically unnatural conditions, which limits the translational potential of preclinical research. Here, we addressed this problem by conducting an unbiased, prospective study of behavioral changes in mice within a natural homecage environment using conventional preclinical pain assays. Unexpectedly, we observed that cage-lid hanging, a species-specific elective behavior, was the only homecage behavior reliably impacted by pain assays. Noxious stimuli reduced hanging behavior in an intensity-dependent manner, and the reduction in hanging could be restored by analgesics. Finally, we developed an automated approach to assess hanging behavior. Collectively, our results indicate that the depression of hanging behavior is a novel, ethologically valid, and translationally relevant pain outcome measure in mice that could facilitate the study of pain and analgesic development.

The Influence of Behavioral, Social, and Environmental Factors on Reproducibility and Replicability in Aquatic Animal Models.

The publication of reproducible, replicable, and translatable data in studies utilizing animal models is a scientific, practical, and ethical necessity. This requires careful planning and execution of experiments and accurate reporting of results. Recognition that numerous developmental, environmental, and test-related factors can affect experimental outcomes is essential for a quality study design. Factors commonly considered when designing studies utilizing aquatic animal species include strain, sex, or age of the animal; water quality; temperature; and acoustic and light conditions. However, in the aquatic environment, it is equally important to consider normal species behavior, group dynamics, stocking density, and environmental complexity, including tank design and structural enrichment. Here, we will outline normal species and social behavior of 2 commonly used aquatic species: zebrafish (Danio rerio) and Xenopus (X. laevis and X. tropicalis). We also provide examples as to how these behaviors and the complexity of the tank environment can influence research results and provide general recommendations to assist with improvement of reproducibility and replicability, particularly as it pertains to behavior and environmental complexity, when utilizing these popular aquatic models.

Author Correction: Evaluating analgesic efficacy and administration route following craniotomy in mice using the grimace scale.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Head Tilt in Immunodeficient Mice Due to Contamination of Drinking Water by Burkholderia gladioli.

Immunodeficient mice in multiple holding rooms presented with head tilt, circling, spinning when picked up by the tail, dehydration, and lethargy. Burkholderia gladioli, a plant pathogen, was identified as the causative agent. Environmental testing revealed the presence of B. gladioli within the automatic watering system, water bottles, and sipper tubes. Here we describe steps taken to reduce the presence of this organism within the automatic watering system and water bottles. Facilities housing immunodeficient mice should take measures to minimize the accumulation of biofilm within their water-supply systems.

Evaluating analgesic efficacy and administration route following craniotomy in mice using the grimace scale.

Most research laboratories abide by guidelines and mandates set by their research institution regarding the administration of analgesics to control pain during the postoperative period. Unfortunately, measuring pain originating from the head is difficult, making adequate decisions regarding pain control following stereotaxic surgery problematic. In addition, most postsurgical analgesia protocols require multiple injections over several days, which may cause stress and distress during a critical recovery period. Here we sought to (1) assess the degree of postoperative pain following craniotomy in mice, (2) compare the efficacy of three common rodent analgesics (carprofen, meloxicam and buprenorphine) for reducing this pain and (3) determine whether the route of administration (injected or self-administered through the drinking supply) influenced pain relief post-craniotomy. Using the mouse grimace scale (MGS), we found that injectable analgesics were significantly more effective at relieving post-craniotomy pain, however, both routes of administration decreased pain scores in the first 24 h postsurgery. Specifically, buprenorphine administered independently of administration route was the most effective at reducing MGS scores, however, female mice showed greater sensitivity to carprofen when administered through the water supply. Although it is necessary to provide laboratory animals with analgesics after an invasive procedure, there remains a gap in the literature regarding the degree of craniotomy-related pain in rodents and the efficacy of alternative routes of administration. Our study highlights the limitations of administering drugs through the drinking supply, even at doses that are considered to be higher than those currently recommended by most research institutions for treating pain of mild to moderate severity.

Humane Endpoints for Guinea Pigs Used for Mycobacterium tuberculosis Vaccine Research.

Guinea pigs are a commonly used model for tuberculosis vaccine research. Loss of body weight is the most frequently described humane endpoint for animals used in these studies. During a chronic study, we noted labored breathing in some tuberculosis-infected guinea pigs. To develop consistent humane endpoints for these guinea pigs, we performed an observational study using multiple clinical signs. A combination of body weight loss, labored breathing, and activity level during handling estimated the time to euthanasia within approximately 7 d. Histologic severity scores of lesions in the cranial or caudal lung lobe (or both) supported clinical endpoints. This study presents humane endpoints for the refinement of studies using guinea pigs in tuberculosis research.

Report of Workshop on Euthanasia for Zebrafish-A Matter of Welfare and Science.

The increasing importance of zebrafish as a biomedical model organism is reflected by the steadily growing number of publications and laboratories working with this species. Regulatory recommendations for euthanasia as issued in Directive 2010/63/EU are, however, based on experience with fish species used for food production and do not take the small size and specific physiology of zebrafish into account. Consequently, the currently recommended methods of euthanasia in the Directive 2010/63/EU are either not applicable or may interfere with research goals. An international workshop was held in Karlsruhe, Germany, March 9, 2017, to discuss and propose alternative methods for euthanasia of zebrafish. The aim was to identify methods that adequately address the physiology of zebrafish and its use as a biomedical research model, follow the principles of the 3Rs (Replacement, Reduction, and Refinement) in animal experimentation and consider animal welfare during anesthesia and euthanasia. The results of the workshop are summarized here in the form of a white paper.

Lidocaine Hydrochloride Compared with MS222 for the Euthanasia of Zebrafish (Danio rerio).

Despite several shortcomings, MS222 is the most commonly used chemical agent for euthanasia of zebrafish. Although lidocaine hydrochloride has some advantages over MS222, its effectiveness as a euthanasia agent for zebrafish is unknown. Larvae at 9 to 16 d postfertilization were exposed to 250 mg/L MS222 or 400, 500, 600, 700, 800, 900, or 1000 mg/L lidocaine and observed for cessation of heartbeat. Adult zebrafish were exposed to 250 mg/L MS222 or 400, 500, or 600 mg/L lidocaine; times to loss of righting reflex, cessation of opercular movement, and complete recovery; body length; aversive behavior; and gross and microscopic evidence of acute toxicity were evaluated. The heartbeat was not lost from any larvae in any group, regardless of drug or dosage. For adults, time to loss of righting reflex was greatest in the 500-mg/L lidocaine group. Opercular movement ceased earlier in all lidocaine groups compared with the MS222 group. Fish in the 500-mg/L lidocaine group were smaller than those in other groups. Fewer fish in the lidocaine groups displayed aversive behavior (erratic swimming and piping) compared with the MS222 group. No fish in the lidocaine hydrochloride groups (n = 30) recovered from euthanasia, whereas one fish in the MS222 group did (n = 10). Neither the MS222 nor lidocaine groups showed any gross or histologic changes suggestive of acute toxicity. Our results suggest that lidocaine hydrochloride may be an effective alternative chemical euthanasia agent for adult zebrafish but should not be used in larval fish.

Restrictions on the Importation of Zebrafish into Canada Associated with Spring Viremia of Carp Virus.

The zebrafish model system is helping researchers improve the health and welfare of people and animals and has become indispensable for advancing biomedical research. As genetic engineering is both resource intensive and time-consuming, sharing successfully developed genetically modified zebrafish lines throughout the international community is critical to research efficiency and to maximizing the millions of dollars in research funding. New restrictions on importation of zebrafish into Canada based on putative susceptibility to infection by the spring viremia of carp virus (SVCV) have been imposed on the scientific community. In this commentary, we review the disease profile of SVCV in fish, discuss the findings of the Canadian government's scientific assessment, how the interpretations of their assessment differ from that of the Canadian research community, and describe the negative impact of these regulations on the Canadian research community and public as it pertains to protecting the health of Canadians.

Recommendations for Health Monitoring and Reporting for Zebrafish Research Facilities.

The presence of subclinical infection or clinical disease in laboratory zebrafish may have a significant impact on research results, animal health and welfare, and transfer of animals between institutions. As use of zebrafish as a model of disease increases, a harmonized method for monitoring and reporting the health status of animals will facilitate the transfer of animals, allow institutions to exclude diseases that may negatively impact their research programs, and improve animal health and welfare. All zebrafish facilities should implement a health monitoring program. In this study, we review important aspects of a health monitoring program, including choice of agents, samples for testing, available testing methodologies, housing and husbandry, cost, test subjects, and a harmonized method for reporting results. Facilities may use these recommendations to implement their own health monitoring program.

The Behavioral Effects of Single Housing and Environmental Enrichment on Adult Zebrafish (Danio rerio).

Environmental enrichment provides laboratory-housed species the opportunity to express natural behavior and exert control over their home environment, thereby minimizing stress. We sought to determine whether providing an artificial plant in the holding tank as enrichment influenced anxiety-like behaviors and place-preference choice in adult zebrafish. Fish were housed singly or in social groups of 5 for 3 wk in 1 of 4 experimental housing environments: single-housed enriched (n = 30), single-housed barren (n = 30), group-housed enriched (n = 30), and group-housed barren (n = 30). On week 4, individual fish were selected randomly from each of the experimental housing environments and tested by using novel-tank, light-dark, and place-preference tests. Housing fish singly in a barren environment increased anxiety-like behaviors in the novel-tank and light-dark behavioral tests. Single-housed zebrafish in barren tanks as well as zebrafish group-housed with conspecifics, both with and without plant enrichment, spent more time associating with conspecifics than with the artificial plant enrichment device during the place-preference test. Single-housed fish maintained in enriched tanks displayed no preference between a compartment with conspecifics or an artificial plant. Our results suggest the addition of an artificial plant as enrichment may benefit single-housed zebrafish when social housing is not possible.

Tolerance and efficacy of emamectin benzoate and ivermectin for the treatment of Pseudocapillaria tomentosa in laboratory zebrafish (Danio rerio).

Tolerance of adult zebrafish and efficacy of emamectin benzoate and ivermectin in eliminating Pseudocapillaria tomentosa infection were evaluated. In the tolerance study, behavioral changes, fecundity, histopathology, and mortality were evaluated for in-feed administration of emamectin (0.05, 0.10, and 0.25 mg/kg) and ivermectin (0.05 and 0.10 mg/kg). All doses of emamectin were well tolerated. Ivermectin 0.05 mg/kg administration resulted in mild behavioral changes and a transient decrease in fecundity. Ivermectin 0.10 mg/kg administration resulted in severe behavioral changes and some mortality. In the efficacy study, emamectin (0.05 and 0.25 mg/kg) and ivermectin (0.05 mg/kg) were evaluated for their efficacy in eliminating P. tomentosa infection. Emamectin reduced parasite burden in infected zebrafish, and ivermectin eliminated intestinal nematode infections. Despite a small margin of safety, ivermectin 0.05 mg/kg was effective at eliminating P. tomentosa infection in adult zebrafish. Higher doses or a longer course of treatment may be needed for complete elimination of P. tomentosa infection using emamectin. In this study, we propose two possible treatments for intestinal nematode infections in zebrafish.

Efficacy and safety of 5 anesthetics in adult zebrafish (Danio rerio).

Although the safety and efficacy of tricaine methanesulfonate (MS222) for anesthesia of fish are well established, other anesthetics used less commonly in fish have been less extensively evaluated. Therefore, we compared gradual cooling, lidocaine hydrochloride (300, 325, and 350 mg/L), metomidate hydrochloride (2, 4, 6, 8, and 10 mg/L), and isoflurane (0.5 mL/L) with MS222 (150 mg/L) for anesthesia of adult zebrafish. The efficacy and safety of each agent was evaluated by observing loss of equilibrium, slowing of opercular movement, response to tail-fin pinch, recovery time, and anesthesia-associated mortality rates. At 15 min after anesthetic recovery, we used a novel-tank test to evaluate whether anesthetic exposure influenced short-term anxiety-like behavior. Behavioral parameters measured included latency to enter and number of transitions to the upper half of the tank, number of erratic movements, and number of freezing bouts. Behavior after anesthesia was unaltered regardless of the anesthetic used. Efficacy and safety differed among the anesthetics evaluated. Gradual cooling was useful for short procedures requiring immobilization only, but all instrumentation and surfaces that come in contact with fish must be maintained at approximately 10 °C. MS222 and lidocaine hydrochloride at 325 mg/L were effective as anesthetic agents for surgical procedures in adult zebrafish, but isoflurane and high-dose lidocaine hydrochloride were unsuitable as sole anesthetic agents due to high (30%) mortality rates. Although MS222 remains the best choice for generating a surgical plane of anesthe- sia, metomidate hydrochloride and gradual cooling were useful for sedation and immobilization for nonpainful procedures.

Evaluation of 5 cleaning and disinfection methods for nets used to collect zebrafish (Danio rerio).

Few standardized methods of cleaning and disinfecting equipment in zebrafish facilities have been published, even though the effectiveness of these procedures is vital to preventing the transmission of pathogenic organisms. Four chemical disinfectants and rinsing with municipal tap water were evaluated for their ability to disinfect nets used to capture zebrafish. The disinfectants included benzalkonium chloride+methylene blue, sodium hypochlorite, chlorine dioxide, and potassium peroxymonosulfate+sodium chloride for a soak time of 5 or 30 min. Disinfection effectiveness was evaluated by using an ATP-based system that measured the reduction in absolute number and percentage of relative light units. In addition, nets were cultured aerobically on blood and MacConkey agar plates to determine the number of bacteria remaining after disinfection procedures. Soaking nets in sodium hypochlorite for 30 min and in potassium peroxymonosulfate+sodium chloride for 5 or 30 min were effective means of disinfection, according to at least 90% reduction in the number of relative light units and no bacterial growth after cleaning. These results will aid facility managers, veterinarians and investigators in selecting net cleaning and disinfection protocols.

Trichodina xenopodus, a ciliated protozoan, in a laboratory-maintained Xenopus laevis.

A postmortem evaluation of a domestically bred, adult, female Xenopus laevis revealed the presence of a urinary bladder protozoan consistent with Trichodina xenopodus. T. xenopodus is considered an incidental finding, as its presence in the urinary bladder in frogs has not been correlated with disease or with urinary bladder epithelial lesions. Trichodina spp. are ciliated protozoa known to colonize many species of amphibians and fish. These protozoa frequently inhabit the skin and gills, but may also be present in the urinary bladder of infected animals. Their presence on the skin and gills in low numbers is not related to disease; however, large numbers may indicate poor water quality and overcrowding.

Gavaging adult zebrafish.

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or agents in solutions for scientific research. Current methods for administering compounds orally to adult zebrafish are inaccurate due to variability in voluntary consumption by the fish. A gavage procedure was developed to deliver precise quantities of infectious agents to zebrafish for study in biomedical research. Adult zebrafish over 6 months of age were anesthetized with 150 mg/L of buffered MS-222 and gavaged with 5 µl of solution using flexible catheter implantation tubing attached to a cut 22-G needle tip. The flexible tubing was lowered into the oral cavity of the zebrafish until the tip of the tubing extended past the gills (approximately 1 cm). The solution was then injected slowly into the intestinal tract. This method was effective 88% of the time, with fish recovering uneventfully. This procedure is also efficient as one person can gavage 20-30 fish in one hour. This method can be used to precisely administer agents for infectious diseases studies, or studies of other compounds in adult zebrafish.