Population genomics of invasive rodents on islands: Genetic consequences of colonization and prospects for localized synthetic gene drive.

Introduced rodent populations pose significant threats worldwide, with particularly severe impacts on islands. Advancements in genome editing have motivated interest in synthetic gene drives that could potentially provide efficient and localized suppression of invasive rodent populations. Application of such technologies will require rigorous population genomic surveys to evaluate population connectivity, taxonomic identification, and to inform design of gene drive localization mechanisms. One proposed approach leverages the predicted shifts in genetic variation that accompany island colonization, wherein founder effects, genetic drift, and island-specific selection are expected to result in locally fixed alleles (LFA) that are variable in neighboring nontarget populations. Engineering of guide RNAs that target LFA may thus yield gene drives that spread within invasive island populations, but would have limited impacts on nontarget populations in the event of an escape. Here we used pooled whole-genome sequencing of invasive mouse (Mus musculus) populations on four islands along with paired putative source populations to test genetic predictions of island colonization and characterize locally fixed Cas9 genomic targets. Patterns of variation across the genome reflected marked reductions in allelic diversity in island populations and moderate to high degrees of differentiation from nearby source populations despite relatively recent colonization. Locally fixed Cas9 sites in female fertility genes were observed in all island populations, including a small number with multiplexing potential. In practice, rigorous sampling of presumptive LFA will be essential to fully assess risk of resistance alleles. These results should serve to guide development of improved, spatially limited gene drive design in future applications.

Genetic Biocontrol for Invasive Species.

Invasive species are increasingly affecting agriculture, food, fisheries, and forestry resources throughout the world. As a result of global trade, invasive species are often introduced into new environments where they become established and cause harm to human health, agriculture, and the environment. Prevention of new introductions is a high priority for addressing the harm caused by invasive species, but unfortunately efforts to prevent new introductions do not address the economic harm that is presently manifested where invasive species have already become established. Genetic biocontrol can be defined as the release of organisms with genetic methods designed to disrupt the reproduction of invasive populations. While these methods offer the potential to control or even eradicate invasive species, there is a need to ensure that genetic biocontrol methods can be deployed in a way that minimizes potential harm to the environment. This review provides an overview of the state of genetic biocontrol, focusing on several approaches that were the subject of presentations at the Genetic Biocontrol for Invasive Species Workshop in Tarragona, Spain, March 31st, 2019, a workshop sponsored by the OECD's Co-operative Research Program on Biological Resource Management for Sustainable Agricultural Systems. The review considers four different approaches to genetic biocontrol for invasive species; sterile-release, YY Males, Trojan Female Technique, and gene drive. The different approaches will be compared with respect to the efficiency each affords as a genetic biocontrol tool, the practical utility and cost/benefits associated with implementation of the approach, and the regulatory considerations that will need to be addressed for each. The opinions expressed and arguments employed in this publication are the sole responsibility of the authors and do not necessarily reflect those of the OECD or of the governments of its Member countries.

Locally Fixed Alleles: A method to localize gene drive to island populations.

Invasive species pose a major threat to biodiversity on islands. While successes have been achieved using traditional removal methods, such as toxicants aimed at rodents, these approaches have limitations and various off-target effects on island ecosystems. Gene drive technologies designed to eliminate a population provide an alternative approach, but the potential for drive-bearing individuals to escape from the target release area and impact populations elsewhere is a major concern. Here we propose the "Locally Fixed Alleles" approach as a novel means for localizing elimination by a drive to an island population that exhibits significant genetic isolation from neighboring populations. Our approach is based on the assumption that in small island populations of rodents, genetic drift will lead to alleles at multiple genomic loci becoming fixed. In contrast, multiple alleles are likely to be maintained in larger populations on mainlands. Utilizing the high degree of genetic specificity achievable using homing drives, for example based on the CRISPR/Cas9 system, our approach aims at employing one or more locally fixed alleles as the target for a gene drive on a particular island. Using mathematical modeling, we explore the feasibility of this approach and the degree of localization that can be achieved. We show that across a wide range of parameter values, escape of the drive to a neighboring population in which the target allele is not fixed will at most lead to modest transient suppression of the non-target population. While the main focus of this paper is on elimination of a rodent pest from an island, we also discuss the utility of the locally fixed allele approach for the goals of population suppression or population replacement. Our analysis also provides a threshold condition for the ability of a gene drive to invade a partially resistant population.

Evaluation of automated and manual DNA purification methods for detecting Ricinus communis DNA during ricin investigations.

In April of 2013, letters addressed to the President of United States and other government officials were intercepted and found to be contaminated with ricin, heightening awareness about the need to evaluate laboratory methods for detecting ricin. This study evaluated commercial DNA purification methods for isolating Ricinus communis DNA as measured by real-time polymerase chain reaction (PCR). Four commercially available DNA purification methods (two automated, MagNA Pure compact and MagNA Pure LC, and two manual, MasterPure complete DNA and RNA purification kit and QIAamp DNA blood mini kit) were evaluated. We compared their ability to purify detectable levels of R. communis DNA from four different sample types, including crude preparations of ricin that could be used for biological crimes or acts of bioterrorism. Castor beans, spiked swabs, and spiked powders were included to simulate sample types typically tested during criminal and public health investigations. Real-time PCR analysis indicated that the QIAamp kit resulted in the greatest sensitivity for ricin preparations; the MasterPure kit performed best with spiked powders. The four methods detected equivalent levels by real-time PCR when castor beans and spiked swabs were used. All four methods yielded DNA free of PCR inhibitors as determined by the use of a PCR inhibition control assay. This study demonstrated that DNA purification methods differ in their ability to purify R. communis DNA; therefore, the purification method used for a given sample type can influence the sensitivity of real-time PCR assays for R. communis.

Ferric uptake regulator-dependent antinitrosative defenses in Salmonella enterica serovar Typhimurium pathogenesis.

Herein we report an important role for the ferric uptake regulator (Fur) in the resistance of Salmonella enterica serovar Typhimurium to the reactive nitrogen species produced by inducible nitric oxide (NO) synthase in an NRAMP1(r) murine model of acute systemic infection. The expression of fur protected Salmonella grown under normoxic and hypoxic conditions against the bacteriostatic activity of NO. The hypersusceptibility of fur-deficient Salmonella to the cytotoxic actions of NO coincides with a marked repression of respiratory activity and the reduced ability of the bacteria to detoxify NO. A fur mutant Salmonella strain contained reduced levels of the terminal quinol oxidases of the electron transport chain. Addition of the heme precursor δ-aminolevulinic acid restored the cytochrome content, respiratory activity, NO consumption, and wild-type growth in bacteria undergoing nitrosative stress. The innate antinitrosative defenses regulated by Fur added to the adaptive response associated with the NO-detoxifying activity of the flavohemoprotein Hmp. Our investigations indicate that, in addition to playing a critical role in iron homeostasis, Fur is an important antinitrosative determinant of Salmonella pathogenesis.

Use of sentinel laboratories by clinicians to evaluate potential bioterrorism and emerging infections.

With the persistent threat of emerging infectious diseases and bioterrorism, it has become increasingly important that clinicians be able to identify the diseases that might signal the occurrence of these unusual events. Essential to a thoughtful diagnostic approach is understanding when to initiate a public health investigation and how to appropriately use commonly performed microbiology procedures in the sentinel laboratory to evaluate potential pathogens. Although diagnostic test development is evolving rapidly, recognizing many of these pathogens continues to challenge the capabilities of most sentinel laboratories. Therefore, effective, ongoing communication and education among clinicians, infection control personnel, sentinel laboratorians, public health authorities, and Laboratory Response Network reference laboratorians is the key to preparedness.

Outbreak of listeriosis among Mexican immigrants as a result of consumption of illicitly produced Mexican-style cheese.

BACKGROUND: In 2000, an outbreak of listeriosis among Hispanic persons was identified in Winston-Salem, North Carolina. The objectives of the present study were to identify the source of, strains associated with, and risk factors for Listeria monocytogenes infection for patients affected by the outbreak. METHODS: Microbiological, case-control, and environmental investigations were conducted. Participants in the case-control study were case patients who became infected with L. monocytogenes between 1 October 2000 and 31 January 2001 and control subjects who were matched with case patients on the basis of ethnicity, sex, age, and pregnancy status. All participants were residents of Winston-Salem. RESULTS: We identified 13 patients, all of whom were Hispanic, including 12 females who were 18-38 years of age. Eleven case patients were pregnant; infection with L. monocytogenes resulted in 5 stillbirths, 3 premature deliveries, and 3 infected newborns. Case patients were more likely than control subjects to have eaten the following foods: fresh, unlabeled, Mexican-style cheese sold by door-to-door vendors (matched odds ratio [MOR], 17.5; 95% confidence interval [CI], 2.0-152.5); queso fresco, a Mexican-style soft cheese (MOR, 7.3; 95% CI, 1.4-37.5); and hot dogs (MOR, 4.6; 95% CI, 1.1-19.4). L. monocytogenes isolates recovered from 10 female case patients, from cheese bought from a door-to-door vendor, from unlabeled cheese from 2 Hispanic markets, and from raw milk from a local dairy had indistinguishable patterns on pulsed-field gel electrophoresis. CONCLUSIONS: This outbreak of listeriosis was caused by noncommercial, fresh, Mexican-style cheese made from contaminated raw milk traced to 1 local dairy. We recommend educating Hispanic women about food safety while they are pregnant, enforcing laws that regulate the sale of raw milk and dairy products made by unlicensed manufacturers, making listeriosis a reportable disease in all states, routinely interviewing case patients, and routinely subtyping clinical L. monocytogenes isolates.