Animal Biosafety Level Research Articles

Effects of Anti-Retroviral Therapy (ART) on Restoration of Lung Immunity and Tuberculosis Reactivation

While the advent of combination antiretroviral therapy (cART, ART) has significantly improved survival, tuberculosis (TB) still accounts for one-third of deaths in Human Immunodeficiency Virus (HIV)-infected populations, suggesting incomplete restoration of immune function by ART. Mtb/Simian Immunodeficiency Virus (SIV) co-infected macaques have been used to model Mtb/HIV co-infection in humans. To identify the components of TB immunity that remain impaired following HIV-infection and ART treatment, macaques with asymptomatic M. tuberculosis (Mtb) infection were subsequently co-infected with SIV. Animals were then randomly assigned to ART or control groups and studied longitudinally. While ART significantly reduced viral loads and increased CD4+T cell counts in whole blood and BAL samples, it did not reduce the relative risk of SIV- induced TB reactivation (RR 1·23, 95% CI 0·97-1·5) in ART treated macaques. CD4+T cells were poorly restored specifically in the lung interstitial region, despite their significant restoration in the alveolar compartment of the lung as well as in the periphery. IDO1 induction on myeloid cells in the iBALT likely contributes to dysregulated T cell homing and impaired lung immunity. Thus, while ART is indispensable for controlling viral replication, CD4+T cells restoration and in preventing opportunistic infection, it is however inadequate in reversing Mtb-specific T cell impairment. This warrants modeling concurrent treatment of TB and HIV to potentially reduce the risk of reactivation of TB due to HIV. The current and future studies like this have the potential to inform treatment strategies in patients with Mtb/HIV co-infection. The study was funded through grants from the National Institute of Health (NIH). Funding Statement: This work was primarily supported by NIH awards to D.K. and J.R. (R01AI111943, R01AI123047) with additional support from NIH awards R01AI111914, R01AI134240, R01AI135726, U19AI111211 and institutional awards P51OD111033 and P51OD111004. Declaration of Interests: The authors declare that no conflict of interest exists. Ethics Approval Statement: All the animals were housed in the Animal Biosafety level III (ABSL3) at Tulane National Primate Research Center where they were treated as per the standards recommended by Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) International and the NIH Guide for the Care and Use of Laboratory Animals. The study was approved by the IACUC of the Tulane National Primate Research Center (protocols P0247R, P0324, P0295R).

Biosafety and biosecurity requirements for Orientia spp. diagnosis and research: recommendations for risk-based biocontainment, work practices and the case for reclassification to risk group 2

Scrub typhus is an important arthropod-borne disease causing significant acute febrile illness by infection with Orientia spp.Using a risk-based approach, this review examines current practice, the evidence base and regulatory requirements regarding matters of biosafety and biosecurity, and presents the case for reclassification from Risk Group 3 to Risk Group 2 along with recommendations for safe working practices of risk-based activities during the manipulation of Orientia spp. in the laboratory.We recommend to reclassify Orientia spp. to Risk Group 2 based on the classification for RG2 pathogens as being moderate individual risk, low community risk. We recommend that low risk activities, can be performed within a biological safety cabinet located in a Biosafety Level (BSL) 2 core laboratory using standard personal protective equipment. But when the risk assessment indicates, such as high concentration and volume, or aerosol generation, then a higher biocontainment level is warranted. For, the majority of animal activities involving Orientia spp., Animal BSL 2 (ABSL2) is recommended however where high risk activities are performed including necropsies, Animal BSL (ABSL3) is recommended.

Validation of the Bio-Response Solutions Human-28 Low-Temperature Alkaline Hydrolysis System.

High temperature alkaline hydrolysis (AH) is recognized as an alternative method for sterilization and disposition of animal carcasses and human remains. The aim of this study is to validate the low temperature (LT) AH process specific to its use in the Bio-Response Solutions, Inc. Human-28 LT System. A 313-lb pig was processed using the manufacturers recommended cycle parameters. Stainless steel sample vials containing spore suspensions of Geobacillus stearothermophilus were implanted into the pig's deep tissue to validate the efficacy of the process conditions. Spore suspensions of Bacillus thuringiensis were suspended in the vessel headspace to validate sterilization. The spore challenge was greater than the recommended 106 log used to determine sterilization. MALDI-TOF mass spectrometry analysis was used to validate the destruction of prion-sized particles in processed effluent. Complete inactivation of spores and digestion of animal tissue were achieved after processing in the Bio-Response Solutions Human-28 LT Alkaline Hydrolysis System. Complete inactivation of spores was achieved when exposed to heat in the animal carcass and headspace. No peptide fragments larger than 2500 Da were observed in the treatment effluent. The Bio-Response Solutions, Inc. Human-28 LT Alkaline Hydrolysis System was as effective as high-temperature alkaline hydrolysis for use on animal and human tissue. LT AH for tissue and bodies exceeded the sterility assurance level III of the US State and Territorial Association on Alternative Treatment Technologies and sterility requirements for animal biosafety level-3 and -4 facilities. LT AH process validated destruction of prion-sized particles.

Validating Autoclave Cycles for Carcass Disposal in Animal Biosafety Level 2/3 Containment Laboratories.

Animal carcasses differ in composition from other types of solid waste, and through prior testing it was determined that cycle parameters applied to general, solid biohazardous waste did not ensure proper sterilization of ferret carcasses. The goals of this study were to develop and validate an autoclave cycle that would ensure the decontamination of infectious animal carcasses before removal from an animal biosafety level 2/3 containment suite for downstream disposal and to test different ways to prepare and package animal carcasses for autoclaving. Intact ferret carcasses were implanted with biological indicators, and the carcasses were placed in biohazard bags, then into metal pans. To test the efficacy of the autoclave cycle on larger biomasses, 1, 2, or 4 ferret carcasses were placed in a biohazard bag. A total of 4 carcasses were placed in each pan. An autoclave cycle was created to begin the study. After initial tests, minor modifications to the initial test cycle parameters were made, and a new cycle was validated for ferret carcasses up to 2 kg each. Parameters for the validated cycle were as follows: sterilization time 240 minutes, temperature 125°C, 5 prevacuum pulses, and chamber pressure 15 psi. The results of this study indicate that an extended sterilization time is required to successfully decontaminate animal carcasses compared with regular, solid, and biohazardous waste. This study demonstrates that it is possible to sterilize multiple intact ferret carcasses per load under validated autoclave cycle conditions.

A biosafety level-2 dose-dependent lethal mouse model of spotted fever rickettsiosis: Rickettsia parkeri Atlantic Rainforest strain.

BackgroundThe species of the Rickettsia genus are separated into four groups: the ancestral group, typhus group, transitional group and spotted fever group. Rickettsia parkeri, a spotted fever group Rickettsia, has been reported across the American continents as infecting several tick species and is associated with a relatively mild human disease characterized by eschar formation at the tick feeding site, regional lymphadenopathy, fever, myalgia and rash. Currently, there are several mouse models that provide good approaches to study the acute lethal disease caused by Rickettsia, but these models can only be performed in an animal biosafety level 3 laboratory. We present an alternative mouse model for acute lethal rickettsial disease, using R. parkeri Atlantic Rainforest strain and C3H/HeN mice, with the advantage that this model can be studied in an animal biosafety level 2 laboratory.Principal findingsIn the C3H/HeN mouse model, we determined that infection with 1x106 and 1x107 viable R. parkeri Atlantic Rainforest strain organisms produced dose-dependent severity, whereas infection with 1x108 viable bacteria resulted in a lethal illness. The animals became moribund on day five or six post-infection. The lethal disease was characterized by ruffled fur, erythema, labored breathing, decreased activity, and hunched posture, which began on day three post-infection (p.i.) and coincided with the peak bacterial loads. Significant splenomegaly (on days three and five p.i.), neutrophilia (on days three and five p.i.), and thrombocytopenia (on days one, three and five p.i.) were observed.SignificanceSince R. parkeri is used at biosafety level 2, the greatest advantage of this inbred mouse model is the ability to investigate immunity and pathogenesis of rickettsiosis with all the tools available at biosafety level 2.

Development of a Model of Porcine Epidemic Diarrhea in Microminipigs.

Porcine epidemic diarrhea virus (PEDV) induces an often fatal gastrointestinal disease in piglets. In this study, we performed a PEDV infection experiment with the Microminipig, the smallest of experimental minipigs, as a novel small animal model. We orally inoculated a neonatal Microminipig with an intestinal homogenate of a PEDV-infected pig and housed it in a small cage originally designed for rats in an animal biosafety level 2 facility. The infected Microminipig showed the typical signs of porcine epidemic diarrhea (PED), such as watery diarrhea, loss of appetite and weight loss. We also recognized a high amount of excreted PEDV in its rectal swabs and villus atrophy of the small intestine. These results suggest that the Microminipig is a good small animal model for PED, which may contribute to a better understanding of the pathogenesis of PEDV.

Biosafety and data quality considerations for animal experiments with highly infectious agents at ABSL-3 facilities

Animal models are crucial for the study of severe infectious diseases, which is essential for determining their pathogenesis and the development of vaccines and drugs. Animal experiments involving risk grade 3 agents such as SARS CoV, HIV, M.tb, H7N9, and Brucella must be conducted in an Animal Biosafety Level 3 (ABSL-3) facility. Because of the in vivo work, the biosafety risk in ABSL-3 facilities is higher than that in BSL-3 facilities. Undoubtedly, management practices must be strengthened to ensure biosafety in the ABSL-3 facility. Meanwhile, we cannot ignore the reliable scientific results obtained from animal experiments conducted in ABSL-3 laboratories. It is of great practical significance to study the overall biosafety concepts that can increase the scientific data quality. Based on the management of animal experiments in the ABSL-3 Laboratory of Wuhan University, combined with relevant international and domestic literature, we indicate the main safety issues and factors affecting animal experiment results at ABSL-3 facilities. Based on these issues, management practices regarding animal experiments in ABSL-3 facilities are proposed, which take into account both biosafety and scientifically sound data.

Laboratory Aerosol Hazard Assessment Utilizing Material Balance Models

Models that are simple to use and interpret and that predict aerosol concentration and integrated exposure dose are needed for local, timely, and cost-effective evaluation of risks associated with biocontainment operations. A material balance model is presented with explicit assumptions that provide a safety context for assessing potential laboratory operating and accident scenarios (1) to support determination of aerosol concentration when limited experimental data are available or (2) to assist in the interpretation of experimental results. The model is used to assess recent experimental data for large animal primary containment systems in animal biosafety level 4; it is also applied to estimate integrated dose from particle concentration for assessments of potential exposure of laboratory workers. The model is further shown to reproduce previously published laboratory accident results.

Validation and Verification of Steam Sterilization Procedures for the Decontamination of Biological Waste in a Biocontainment Laboratory

One of the most critical aspects of safe operations in biosafety level 3 and 4 and animal biosafety level 3 and 4 biocontainment laboratories is the effective inactivation of biological select agen...

Performance Characteristics of a Primary Containment System for Large Animals in Animal Biosafety Level 4

Flexible plastic-walled HEPA-filtered containment chambers, referred to as “primary containment systems” in the Biosafety in Microbiological and Biomedical Laboratories, have been used in animal biosafety level 4 (ABSL-4) in the past as a means to control environmental contamination at its source. The air-cleaning efficiency of these systems, including particle decay rates and protection factors for large animal care staff, is understudied in the literature. A series of studies using large animals (sheep) as well as aerosolized salt solutions were conducted to quantify the performance characteristics of a commercially available primary containment system in an ABSL-4 room (operating at biosafety level 0). When charged with particles that closely match the particle size distribution of loose-housed sheep, results show that particle counts inside the primary containment system are >45 000 times higher than particles outside the chamber in ABSL-4. These data demonstrate that primary containment systems of this type can provide significant safety enhancements and greater contamination control for ABSL-4 staff who work with loose-housed animals.

Detection of Ebola Virus RNA Through Aerosol Sampling of Animal Biosafety Level 4 Rooms Housing Challenged Nonhuman Primates.

Ebola virus disease is a serious illness of humans and nonhuman primates (NHPs). Direct contact has been shown to be the primary source of Ebola (EBOV) transmission. We used a high-volume air sampler to determine whether EBOV could be detected during 3 independent studies with EBOV-challenged NHPs. Viral RNA was recovered during days 9 and 10 of Study I and days 7 and 8 of Study III. Viral RNA levels were below limits of detection during all other collections. The results demonstrate that the biosafety level 4 (BSL-4) suit protects workers from aerosols in a BSL-4 environment using proper engineering and administrative controls.

Comparison of respiratory inductive plethysmography versus head-out plethysmography for anesthetized nonhuman primates in an animal biosafety level 4 facility

For inhalational studies and aerosol exposures to viruses, head-out plethysmography acquisition has been traditionally used for the determination of estimated inhaled dose in anesthetized nonhuman primates prior to or during an aerosol exposure. A pressure drop across a pneumotachograph is measured within a sealed chamber during inspiration/exhalation of the nonhuman primate, generating respiratory values and breathing frequencies. Due to the fluctuation of depth of anesthesia, pre-exposure respiratory values can be variable, leading to less precise and accurate dosing calculations downstream. Although an anesthesia infusion pump may help stabilize the depth of sedation, pumps are difficult to use within a sealed head-out plethysmography chamber. Real-time, head-out plethysmography acquisition could increase precision and accuracy of the measurements, but the bulky equipment needed for head-out plethysmography precludes real-time use inside a Class III biological safety cabinet, where most aerosol exposures occur. However, the respiratory inductive plethysmography (RIP) acquisition method measures the same respiratory parameters by detecting movement of the chest and abdomen during breathing using two elastic bands within the Class III biological safety cabinet. As respiratory values are relayed to a computer for software integration and analysis real-time, adjustment of aerosol exposure duration is based on the depth of sedation of the animal. The objective of this study was to compare values obtained using two methodologies (pre-exposure head-out plethysmography and real-time RIP). Transitioning to RIP technology with real-time acquisition provides more consistent, precise, and accurate aerosol dosing by reducing reported errors in respiratory values from anesthesia variability when using pre-exposure head-out plethysmography acquisition.

October 2016: This Month in JoVE

Here's a look at what's coming up in the October 2016 issue of JoVE: The Journal of Visualized Experiments. In Immunology and Infection, we showcase a series of four videos detailing safety and logistical procedures for working in an Animal Biosafety Level 4, or (A)BSL-4, laboratory. The first of this group, filmed at the NIH Integrated Research Facility at Fort Detrick, demonstrates the safe entry and exit procedures for work inside an (A)BSL-4 suit laboratory suite. Inhalation studies of high-consequence pathogens can simulate natural aerosol transmission, or allow researchers to investigate outcomes of intentional pathogenic aerosol releases. In the second of this series, the authors walk through the safe operation of aerobiology chambers for maximum containment level pathogens, such as the Ebola virus. The challenge of carrying out medical imaging in a high biosafety environment is the focus of the third of these releases. Here, our Authors detail how to prepare animals infected with high-consequence pathogens for noninvasive medical imaging, whilst ensuring that the equipment remains easily accessed and free from contamination. In the final video of this miniseries, we take an in-depth look at the extra precautions and procedures involved in performing viral assays in a Class II biosafety cabinet in a BSL-4 environment. As a whole, these four releases are a valuable library for researchers handling challenging and potentially harmful biological materials. Woody plants and secondary tree stems are key habitats, as well as being of great cultural and commercial importance. Understanding stem growth and wood formation is therefore an important topic for tree production, conservation, and preservation. In JoVE Genetics this month, Spokevicius et al (our Authors) describe a method to create transgenic somatic tissue sectors directly in the living secondary stem of woody plants. This versatile method can facilitate rapid functional characterization of genes of interest, be utilized in a range of tree species, and test multiple genes and promoters at a high throughput level. Arguably one of the most recognizable paintings of all time, the Mona Lisa is famous for the subject's elusive and indefinable expression. In JoVE Behavior, Soranzo and Newbury (our Authors) demonstrate how a similar visual illusion-dubbed the uncatchable smile because of it's tendency to disappear as the observer tries to catch it-was discovered in La Bella Principessa, also by Da Vinci. Using a combination of methods including interobservation, psychophysical experiments, and structured interviews, the authors reveal that the ambiguity in La Bella Principessa's expression is attributed to a visual illusion at the mouth of the subject similar to that observed in the Mona Lisa. You've just had a sneak peek of the October 2016 issue of JoVE. Visit the website to see the full-length articles, plus many more, in JoVE: The Journal of Visualized Experiments.

Safety Precautions and Operating Procedures in an (A)BSL-4 Laboratory: 4. Medical Imaging Procedures.

Medical imaging using animal models for human diseases has been utilized for decades; however, until recently, medical imaging of diseases induced by high-consequence pathogens has not been possible. In 2014, the National Institutes of Health, National Institute of Allergy and Infectious Diseases, Integrated Research Facility at Fort Detrick opened an Animal Biosafety Level 4 (ABSL-4) facility to assess the clinical course and pathology of infectious diseases in experimentally infected animals. Multiple imaging modalities including computed tomography (CT), magnetic resonance imaging, positron emission tomography, and single photon emission computed tomography are available to researchers for these evaluations. The focus of this article is to describe the workflow for safely obtaining a CT image of a live guinea pig in an ABSL-4 facility. These procedures include animal handling, anesthesia, and preparing and monitoring the animal until recovery from sedation. We will also discuss preparing the imaging equipment, performing quality checks, communication methods from "hot side" (containing pathogens) to "cold side," and moving the animal from the holding room to the imaging suite.

Validating Waste Management Equipment in an Animal Biosafety Level 3 Facility

The Duke-NUS Medical School’s animal biosafety level 3 facility is fitted with 3 types of equipment for waste decontamination: autoclaves, a tissue digester, and an effluent decontamination system ...

The Main Biological Hazards in Animal Biosafety Level 2 Facilities and Strategies for Control

Concern about the biological hazards involved in microbiological research, especially research involving laboratory animals, has increased in recent years. Working in an animal biosafety level 2 facility (ABSL-2), commonly used for research on infectious diseases, poses various biological hazards. Here, the regulations and standards related to laboratory biosafety in China are introduced, the potential biological hazards present in ABSL-2 facilities are analyzed, and a series of strategies to control the hazards are presented.

Novel Approach for Validating Autoclave Cycles for Biomass in BSL-3/-4

The National Biodefense Analysis and Countermeasures Center (NBACC) conducts research at Animal Biosafety Level 3 (ABSL-3) and ABSL-4. At NBACC, all solid waste in ABSL-3 and ABSL-4 must be sterili...

Generation and characterization of large-particle aerosols using a center flow tangential aerosol generator with a non-human-primate, head-only aerosol chamber

Aerosol droplets or particles produced from infected respiratory secretions have the potential to infect another host through inhalation. These respiratory particles can be polydisperse and range from 0.05 to 500 µm in diameter. Animal models of infection are generally established to facilitate the potential licensure of candidate prophylactics and/or therapeutics. Consequently, aerosol-based animal infection models are needed to properly study and counter airborne infections. Ideally, experimental aerosol exposure should reliably result in animal disease that faithfully reproduces the modeled human disease. Few studies have been performed to explore the relationship between exposure particle size and induced disease course for infectious aerosol particles. The center flow tangential aerosol generator (CenTAG™) produces large-particle aerosols capable of safely delivering a variety of infectious aerosols to non-human primates (NHPs) within a Class III Biological Safety Cabinet (BSC) for establishment or refinement of NHP infectious disease models. Here, we report the adaptation of this technology to the Animal Biosafety Level 4 (ABSL-4) environment for the future study of high-consequence viral pathogens and the characterization of CenTAG™-created sham (no animal, no virus) aerosols using a variety of viral growth media and media supplements.

Inhibitory effects of the ultrasound-targeted microbubble destruction-mediated herpes simplex virus-thymidine kinase/ganciclovir system on ovarian cancer in mice.

The aim of the present study was to investigate the effect of the ultrasound-targeted microbubble destruction mediated (UTMD) herpes simplex virus-thymidine kinase (HSV-TK) and ganciclovir (GCV) system on ovarian cancer (OC). This study was conducted between June and December 2012 in the Animal Biosafety Level III Laboratory of Wuhan University. Mice with OC were randomly divided into four groups: i) HSV-TK plus microbubbles (MBs) plus ultrasound (US) (n=15); ii) HSV-TK plus US (n=15); iii) HSV-TK (n=15); and iv) phosphate-buffered saline (n=15). The inhibitory effect and survival time in the experimental groups were compared with those in the control group. The TK protein expression was detected by western blot analysis. Tumor cell apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling and caspase-3 activity analysis. The data showed that the efficiency of HSV-TK gene transfection and the tumor inhibitory effects were significantly increased in the HSV-TK plus MBs plus US group compared with those in the control group (P<0.01). UTMD-mediated HSV-TK treatment has also improved the rat survival rate (P<0.01). In conclusion, UTMD can effectively transfect the HSV-TK gene into target tissues and exert a significant inhibitory effect on OC in mice.

Development of novel mechanisms for housing, handling, and remote monitoring of common marmosets at animal biosafety level 3

The use of common marmosets as an alternative non-human primate model for infectious disease research using BSL-3 viruses such as Rift Valley fever virus (RVFV) presents unique challenges with respect to housing, handling, and safety. Subject matter experts from veterinary care, animal husbandry, biosafety, engineering, and research were consulted to design a pilot experiment using marmosets infected with RVFV. This paper reviews the caging, handling, and safety-related adaptations and modifications that were required to humanely utilize marmosets as a model for high-hazard BSL-3 viral diseases.