Burkholderia pseudomallei JW270 Is Lethal in the Madagascar Hissing Cockroach Infection Model and Can Be Utilized at Biosafety Level 2 to Identify Putative Virulence Factors.

Burkholderia pseudomallei, the causative agent of melioidosis, is classified by the CDC as a tier 1 select agent, and work involving it must be performed in a biosafety level 3 (BSL-3) laboratory. Three BSL-2 surrogate strains derived from B. pseudomallei 1026b, a virulent clinical isolate, have been removed from the CDC select agent list. These strains, Bp82, B0011, and JW270, are highly attenuated in rodent models of melioidosis and cannot be utilized to identify virulence determinants because of their high 50% lethal dose (LD50). We previously demonstrated that the Madagascar hissing cockroach (MHC) is a tractable surrogate host to study the innate immune response against Burkholderia. In this study, we found that JW270 maintains its virulence in MHCs. This surprising result indicates that it may be possible to identify potential virulence genes in JW270 by using MHCs at BSL-2. We tested this hypothesis by constructing JW270 mutations in genes that are required (hcp1) or dispensable (hcp2) for B. pseudomallei virulence in rodents. JW270 Δhcp1 was avirulent in MHCs and JW270 Δhcp2 was virulent, suggesting that MHCs can be used at BSL-2 for the discovery of important virulence factors. JW270 ΔBPSS2185, a strain harboring a mutation in a type IV pilin locus (TFP8) required for full virulence in BALB/c mice, was also found to be attenuated in MHCs. Finally, we demonstrate that the hmqA-G locus, which encodes the production of a family of secondary metabolites called 4-hydroxy-3-methyl-2-alkylquinolines, is important for JW270 virulence in MHCs and may represent a novel virulence determinant.

Genome Resource: Ralstonia solanacearum Phylotype II Sequevar 1 (Race 3 Biovar 2) Strain UW848 From the 2020 U.S. Geranium Introduction.

Ralstonia solanacearum phylotype II sequevar 1 (RsII-1, formerly race 3 biovar 2) causes tomato bacterial wilt, potato brown rot, and Southern wilt of geranium. Strains in RsII-1 cause wilting in potato and tomato at cooler temperatures than tropical lowland R. solanacearum strains. Although periodically introduced, RsII-1 has not established in the United States. This pathogen is of quarantine concern and listed as a Federal Select Agent. We report a rapidly sequenced (<2 days) draft genome of UW848, a RsII-1 isolate introduced to the United States in geranium cuttings in spring 2020. UW848 belongs to the near-clonal cluster of RsII-1 global pandemic strains.

Early detection of viable Francisella tularensis in environmental matrices by culture-based PCR.

BACKGROUND: Francisella tularensis is a fastidious, Gram-negative coccobacillus and is the causative agent of tularemia. To assess viability yet overcome lengthy incubation periods, a culture-based PCR method was used to detect early growth of the lowest possible number of F. tularensis cells. This method utilized a previously developed enhanced F. tularensis growth medium and is based on the change in PCR cycle threshold at the start and end of each incubation. RESULTS: To test method robustness, a virulent Type A1 (Schu4) and B (IN99) strain and the avirulent Live Vaccine Strain (LVS) were incubated with inactivated target cells, humic acid, drinking and well water, and test dust at targeted starting concentrations of 1, 10, and 100 CFU mL- 1 (low, mid, and high, respectively). After 48 h, LVS growth was detected at all targeted concentrations in the presence of 106 inactivated LVS cells; while Schu4 and IN99 growth was detected in the presence of 104 Schu4 or IN99 inactivated cells at the mid and high targets. Early detection of F. tularensis growth was strain and concentration dependent in the presence of fast-growing well water and test dust organisms. In contrast, growth was detected at each targeted concentration by 24 h in humic acid and drinking water for all strains. CONCLUSIONS: Results indicated that the culture-based PCR assay is quick, sensitive, and specific while still utilizing growth as a measure of pathogen viability. This method can circumvent lengthy incubations required for Francisella identification, especially when swift answers are needed during epidemiological investigations, remediation efforts, and decontamination verification.

Avirulent Bacillus anthracis Strain with Molecular Assay Targets as Surrogate for Irradiation-Inactivated Virulent Spores.

The revelation in May 2015 of the shipment of γ irradiation-inactivated wild-type Bacillus anthracis spore preparations containing a small number of live spores raised concern about the safety and security of these materials. The finding also raised doubts about the validity of the protocols and procedures used to prepare them. Such inactivated reference materials were used as positive controls in assays to detect suspected B. anthracis in samples because live agent cannot be shipped for use in field settings, in improvement of currently deployed detection methods or development of new methods, or for quality assurance and training activities. Hence, risk-mitigated B. anthracis strains are needed to fulfill these requirements. We constructed a genetically inactivated or attenuated strain containing relevant molecular assay targets and tested to compare assay performance using this strain to the historical data obtained using irradiation-inactivated virulent spores.

A Standard Method To Inactivate Bacillus anthracis Spores to Sterility via Gamma Irradiation.

In 2015, a laboratory of the United States Department of Defense (DoD) inadvertently shipped preparations of gamma-irradiated spores of Bacillus anthracis that contained live spores. In response, a systematic evidence-based method for preparing, concentrating, irradiating, and verifying the inactivation of spore materials was developed. We demonstrate the consistency of spore preparations across multiple biological replicates and show that two different DoD institutions independently obtained comparable dose-inactivation curves for a monodisperse suspension of B. anthracis spores containing 3 × 1010 CFU. Spore preparations from three different institutions and three strain backgrounds yielded similar decimal reduction (D10) values and irradiation doses required to ensure sterility (DSAL) to the point at which the probability of detecting a viable spore is 10-6 Furthermore, spores of a genetically tagged strain of B. anthracis strain Sterne were used to show that high densities of dead spores suppress the recovery of viable spores. Together, we present an integrated method for preparing, irradiating, and verifying the inactivation of spores of B. anthracis for use as standard reagents for testing and evaluating detection and diagnostic devices and techniques.IMPORTANCE The inadvertent shipment by a U.S. Department of Defense (DoD) laboratory of live Bacillus anthracis (anthrax) spores to U.S. and international destinations revealed the need to standardize inactivation methods for materials derived from biological select agents and toxins (BSAT) and for the development of evidence-based methods to prevent the recurrence of such an event. Following a retrospective analysis of the procedures previously employed to generate inactivated B. anthracis spores, a study was commissioned by the DoD to provide data required to support the production of inactivated spores for the biodefense community. The results of this work are presented in this publication, which details the method by which spores can be prepared, irradiated, and tested, such that the chance of finding residual living spores in any given preparation is 1/1,000,000. These irradiated spores are used to test equipment and methods for the detection of agents of biological warfare and bioterrorism.

Comprehensive Assessment of Inactivation Methods for Madariaga Virus.

The Eastern Equine Encephalitis Virus (EEEV) is an emerging public health threat, with the number of reported cases in the US increasing in recent years. EEEV is a BSL3 pathogen, and the North American strain is a US Federal Select Agent (SA). These restrictions make experiments with EEEV difficult to perform, as high-tech equipment is often unavailable in BSL3 spaces and due to concerns about generating aerosols during manipulations. Therefore, a range of inactivation methods suitable for different downstream analysis methods are essential for advancing research on EEEV. We used heat, chemical, and ultraviolet (UV)-based methods for the inactivation of infected cells and supernatants infected with the non-select agent Madariaga virus (MADV). Although the MADV and EEEV strains are genetically distinct, differing by 8-11% at the amino acid level, they are expected to be similarly susceptible to various inactivation methods. We determined the following to be effective methods of inactivation: heat, TRIzol LS, 4% PFA, 10% formalin, and UV radiation for infected supernatants; TRIzol, 2.5% SDS with BME, 0.2% NP40, 4% PFA, and 10% formalin for infected cells. Our results have the potential to expand the types and complexity of experiments and analyses performed by EEEV researchers.

Select Agent Regulatory Challenges in a Patient Care Setting: Review and Recommendations.

The Federal Select Agent Program ensures the safe and secure possession, use, and transfer of biological select agents and toxins through the select agent regulations (42 CFR §73, 7 CFR §331, and 9 CFR §121). These regulations are primarily written for interpretation by diagnostic and research laboratories, with limited text pertaining to the care of individuals infected with a select agent. The regulations applicable to patient care settings are ambiguous, resulting in challenges with regulatory compliance. The COVID-19 pandemic called attention to these shortcomings and the need to clarify and modify the select agent regulations. In this article, we discuss 3 select agent regulation phrases regarding patient care that need clarification-specifically, the window of time to transfer, patient care setting, and conclusion of patient care-and provide recommendations for improvement. These recommendations include implementing minimum security standards to safeguard patient specimens against theft, loss, or release prior to the appropriate transfer or destruction of the material and increasing the time allowed for the transfer or destruction of specimens before entities are subject to the select agent regulations. We encourage the Federal Select Agent Program to release a policy statement clarifying the select agent regulations regarding patient care discussed herein and to lengthen the designated time to destroy or transfer agents to a registered entity. Addressing these challenges will aid in compliance with the select agent regulations in patient care settings.

LVS ΔcapB-vectored multiantigenic melioidosis vaccines protect against lethal respiratory Burkholderia pseudomallei challenge in highly sensitive BALB/c mice.

Melioidosis, caused by the intracellular bacterial pathogen and Tier 1 select agent Burkholderia pseudomallei (Bp), is a highly fatal disease endemic in tropical areas. No licensed vaccine against melioidosis exists. In preclinical vaccine studies, demonstrating protection against respiratory infection in the highly sensitive BALB/c mouse has been especially challenging. To address this challenge, we have used a safe yet potent live attenuated platform vector, LVS ΔcapB, previously used successfully to develop vaccines against the Tier 1 select agents of tularemia, anthrax, and plague, to develop a melioidosis vaccine. We have engineered melioidosis vaccines (rLVS ΔcapB/Bp) expressing multiple immunoprotective Bp antigens among type VI secretion system proteins Hcp1, Hcp2, and Hcp6, and membrane protein LolC. Administered intradermally, rLVS ΔcapB/Bp vaccines strongly protect highly sensitive BALB/c mice against lethal respiratory Bp challenge, but protection is overwhelmed at very high challenge doses. In contrast, administered intranasally, rLVS ΔcapB/Bp vaccines remain strongly protective against even very high challenge doses. Under some conditions, the LVS ΔcapB vector itself provides significant protection against Bp challenge, and consistent with this, both the vector and vaccines induce humoral immune responses to Bp antigens. Three-antigen vaccines expressing Hcp6-Hcp1-Hcp2 or Hcp6-Hcp1-LolC are among the most potent and provide long-term protection and protection even with a single intranasal immunization. Protection via the intranasal route was either comparable to or statistically significantly better than the single-deletional Bp mutant Bp82, which served as a positive control. Thus, rLVS ΔcapB/Bp vaccines are exceptionally promising safe and potent melioidosis vaccines. IMPORTANCE: Melioidosis, a major neglected disease caused by the intracellular bacterial pathogen Burkholderia pseudomallei, is endemic in many tropical areas of the world and causes an estimated 165,000 cases and 89,000 deaths in humans annually. Moreover, B. pseudomallei is categorized as a Tier 1 select agent of bioterrorism, largely because inhalation of low doses can cause rapidly fatal pneumonia. No licensed vaccine is available to prevent melioidosis. Here, we describe a safe and potent melioidosis vaccine that protects against lethal respiratory challenge with B. pseudomallei in a highly sensitive small animal model-even a single immunization is highly protective, and the vaccine gives long-term protection. The vaccine utilizes a highly attenuated replicating intracellular bacterium as a vector to express multiple key proteins of B. pseudomallei; this vector platform has previously been used successfully to develop potent vaccines against other Tier 1 select agent diseases including tularemia, anthrax, and plague.

Practical Considerations for Navigating Withdrawal from the Federal Select Agent Program.

Introduction: Clear guidance is provided by the Federal Select Agent Program (FSAP) to assist registered entities in nearly all facets of compliance with the Federal select agent regulations (7 CFR Part 331; 9 CFR Part 121; 42 CFR Part 73). If a registered entity chooses to discontinue its registration, detailed instructions for registration withdrawal are deeply embedded within a document entitled "eFSAP Form 1 Amendment Instructions," which is found on the FSAP website within the electronic Federal Select Agent Program (eFSAP) Resource Center. Methods: Using the information found within the eFSAP Form 1 Amendment Instructions, as well as extensive written and verbal guidance provided by the lead assigned entity point of contact at the FSAP, we completed the FSAP withdrawal process during a 12-month period between 2022 and 2023. Discussion: This commentary shares our recent professional experiences navigating the FSAP withdrawal process at the University of Texas Health Science Center at Houston (UTHealth Houston). Successes, challenges, and lessons learned are shared so that others planning or considering withdrawing may benefit from our experience. Conclusion: The resources provided for withdrawal within the eFSAP Form 1 Amendment Instructions are relatively basic, and additional details are not currently found in other FSAP guidance documents. Therefore, direct communication and support from the FSAP to the entity Responsible Officials are imperative to ensure a safe, secure, and compliant withdrawal.

Evaluation of Inactivation Methods for Rift Valley Fever Virus in Mouse Microglia.

Rift Valley fever phlebovirus (RVFV) is a highly pathogenic mosquito-borne virus with bioweapon potential due to its ability to be spread by aerosol transmission. Neurological symptoms are among the worst outcomes of infection, and understanding of pathogenesis mechanisms within the brain is limited. RVFV is classified as an overlap select agent by the CDC and USDA; therefore, experiments involving fully virulent strains of virus are tightly regulated. Here, we present two methods for inactivation of live virus within samples derived from mouse microglia cells using commercially available kits for the preparation of cells for flow cytometry and RNA extraction. Using the flow cytometry protocol, we demonstrate key differences in the response of primary murine microglia to infection with fully virulent versus attenuated RVFV.

Application of multi-criteria decision analysis techniques and decision support framework for informing select agent designation for agricultural animal pathogens.

The United States Department of Agriculture (USDA), Division of Agricultural Select Agents and Toxins (DASAT) established a list of biological agents and toxins (Select Agent List) that potentially threaten agricultural health and safety, the procedures governing the transfer of those agents, and training requirements for entities working with them. Every 2 years the USDA DASAT reviews the Select Agent List, using subject matter experts (SMEs) to perform an assessment and rank the agents. To assist the USDA DASAT biennial review process, we explored the applicability of multi-criteria decision analysis (MCDA) techniques and a Decision Support Framework (DSF) in a logic tree format to identify pathogens for consideration as select agents, applying the approach broadly to include non-select agents to evaluate its robustness and generality. We conducted a literature review of 41 pathogens against 21 criteria for assessing agricultural threat, economic impact, and bioterrorism risk and documented the findings to support this assessment. The most prominent data gaps were those for aerosol stability and animal infectious dose by inhalation and ingestion routes. Technical review of published data and associated scoring recommendations by pathogen-specific SMEs was found to be critical for accuracy, particularly for pathogens with very few known cases, or where proxy data (e.g., from animal models or similar organisms) were used to address data gaps. The MCDA analysis supported the intuitive sense that select agents should rank high on the relative risk scale when considering agricultural health consequences of a bioterrorism attack. However, comparing select agents with non-select agents indicated that there was not a clean break in scores to suggest thresholds for designating select agents, requiring subject matter expertise collectively to establish which analytical results were in good agreement to support the intended purpose in designating select agents. The DSF utilized a logic tree approach to identify pathogens that are of sufficiently low concern that they can be ruled out from consideration as a select agent. In contrast to the MCDA approach, the DSF rules out a pathogen if it fails to meet even one criteria threshold. Both the MCDA and DSF approaches arrived at similar conclusions, suggesting the value of employing the two analytical approaches to add robustness for decision making.

Validating Methods To Eradicate Plant-Pathogenic Ralstonia Strains Reveals that Growth In Planta Increases Bacterial Stress Tolerance.

Plant-pathogenic bacteria in the Ralstonia solanacearum species complex (RSSC) cause highly destructive bacterial wilt disease of diverse crops. Wilt disease prevention and management is difficult because RSSC persists in soil, water, and plant material. Growers need practical methods to kill these pathogens in irrigation water, a common source of disease outbreaks. Additionally, the R. solanacearum race 3 biovar 2 (R3bv2) subgroup is a quarantine pest in many countries and a highly regulated select agent pathogen in the United States. Plant protection officials and researchers need validated protocols to eradicate R3bv2 for regulatory compliance. To meet these needs, we measured the survival of four R3bv2 and three phylotype I RSSC strains following treatment with hydrogen peroxide, stabilized hydrogen peroxide (Huwa-San), active chlorine, heat, UV radiation, and desiccation. No surviving RSSC cells were detected after cultured bacteria were exposed for 10 min to 400 ppm hydrogen peroxide, 50 ppm Huwa-San, 50 ppm active chlorine, or temperatures above 50°C. RSSC cells on agar plates were eradicated by 30 s of UV irradiation and killed by desiccation on most biotic and all abiotic surfaces tested. RSSC bacteria did not survive the cell lysis steps of four nucleic acid extraction protocols. However, bacteria in planta were more difficult to kill. Stems of infected tomato plants contained a subpopulation of bacteria with increased tolerance of heat and UV light, but not oxidative stress. This result has significant management implications. We demonstrate the utility of these protocols for compliance with select agent research regulations and for management of a bacterial wilt outbreak in the field. IMPORTANCE Bacteria in the Ralstonia solanacearum species complex (RSSC) are globally distributed and cause destructive vascular wilt diseases of many high-value crops. These aggressive pathogens spread in diseased plant material and via contaminated soil, tools, and irrigation water. A subgroup of the RSSC, race 3 biovar 2, is a European and Canadian quarantine pathogen and a U.S. select agent subject to stringent and constantly evolving regulations intended to prevent pathogen introduction or release. We validated eradication and inactivation methods that can be used by (i) growers seeking to disinfest water and manage bacterial wilt disease outbreaks, (ii) researchers who must remain in compliance with regulations, and (iii) regulators who are expected to define containment practices. Relevant to all these stakeholders, we show that while cultured RSSC cells are sensitive to relatively low levels of oxidative chemicals, desiccation, and heat, more aggressive treatment, such as autoclaving or incineration, is required to eradicate plant-pathogenic Ralstonia growing inside plant material.

Methods for successful inactivation of Rift Valley fever virus in infected mosquitoes.

Ensuring the successful inactivation of select agent material is critical for maintaining compliance with federal regulations and safeguarding laboratory personnel from exposure to dangerous pathogens. Rift Valley fever virus (RVFV), naturally transmitted by mosquitoes, is classified as a select agent by the CDC and USDA due to its potential to cause significant economic losses to the livestock industry and its demonstrated potential to emerge into naïve geographic areas. Herein we describe several effective inactivation procedures for RVFV infected mosquito samples. We also demonstrate the vaccine strain MP-12 can be used as an appropriate analog for inactivation testing and describe a method of validating inactivation using Amicon filters. Briefly, we show the following inactivation methods are all effective at inactivating RVFV and MP-12 by following the manufacturers'/established protocols: 4 % paraformaldehyde, Trizol LS (ThermoFisher Scientific), MagMAX™-96 Viral RNA Isolation Kit (ThermoFisher Scientific), and Mag-Bind® Viral DNA/RNA 96 Kit (Omega Bio-Tek).