Leveraging a natural murine meiotic drive to suppress invasive populations.

Invasive rodents are a major cause of environmental damage and biodiversity loss, particularly on islands. Unlike insects, genetic biocontrol strategies including population-suppressing gene drives with biased inheritance have not been developed in mice. Here, we demonstrate a gene drive strategy (tCRISPR) that leverages super-Mendelian transmission of the t haplotype to spread inactivating mutations in a haplosufficient female fertility gene (Prl). Using spatially explicit individual-based in silico modeling, we show that tCRISPR can eradicate island populations under a range of realistic field-based parameter values. We also engineer transgenic tCRISPR mice that, crucially, exhibit biased transmission of the modified t haplotype and Prl mutations at levels our modeling predicts would be sufficient for eradication. This is an example of a feasible gene drive system for invasive alien rodent population control.

Characterizing feral swine movement across the contiguous United States using neural networks and genetic data.

Globalization has led to the frequent movement of species out of their native habitat. Some of these species become highly invasive and capable of profoundly altering invaded ecosystems. Feral swine (Sus scrofa × domesticus) are recognized as being among the most destructive invasive species, with populations established on all continents except Antarctica. Within the United States (US), feral swine are responsible for extensive crop damage, the destruction of native ecosystems, and the spread of disease. Purposeful human-mediated movement of feral swine has contributed to their rapid range expansion over the past 30 years. Patterns of deliberate introduction of feral swine have not been well described as populations may be established or augmented through small, undocumented releases. By leveraging an extensive genomic database of 18,789 samples genotyped at 35,141 single nucleotide polymorphisms (SNPs), we used deep neural networks to identify translocated feral swine across the contiguous US. We classified 20% (3364/16,774) of sampled animals as having been translocated and described general patterns of translocation using measures of centrality in a network analysis. These findings unveil extensive movement of feral swine well beyond their dispersal capabilities, including individuals with predicted origins >1000 km away from their sampling locations. Our study provides insight into the patterns of human-mediated movement of feral swine across the US and from Canada to the northern areas of the US. Further, our study validates the use of neural networks for studying the spread of invasive species.

Natural selection on feralization genes contributed to the invasive spread of wild pigs throughout the United States.

Despite a long presence in the contiguous United States (US), the distribution of invasive wild pigs (Sus scrofa × domesticus) has expanded rapidly since the 1980s, suggesting a more recent evolutionary shift towards greater invasiveness. Contemporary populations of wild pigs represent exoferal hybrid descendants of domestic pigs and European wild boar, with such hybridization expected to enrich genetic diversity and increase the adaptive potential of populations. Our objective was to characterize how genetic enrichment through hybridization increases the invasiveness of populations by identifying signals of selection and the ancestral origins of selected loci. Our study focused on invasive wild pigs within Great Smoky Mountains National Park, which represents a hybrid population descendent from the admixture of established populations of feral pigs and an introduction of European wild boar to North America. Accordingly, we genotyped 881 wild pigs with multiple high-density single-nucleotide polymorphism (SNP) arrays. We found 233 markers under putative selection spread over 79 regions across 16 out of 18 autosomes, which contained genes involved in traits affecting feralization. Among these, genes were found to be related to skull formation and neurogenesis, with two genes, TYRP1 and TYR, also encoding for crucial melanogenesis enzymes. The most common haplotypes associated with regions under selection for the Great Smoky Mountains population were also common among other populations throughout the region, indicating a key role of putatively selective variants in the fitness of invasive populations. Interestingly, many of these haplotypes were absent among European wild boar reference genotypes, indicating feralization through genetic adaptation.

Ancestry and genome-wide association study of domestic pigs that survive African swine fever in Uganda.

African swine fever (ASF) is endemic to Uganda and causes annual outbreaks. Some pigs survive these outbreaks and remain asymptomatic but are African swine fever virus (ASFV) positive. The potential heritability and genetic disparities in disease susceptibility among Ugandan pigs are not fully understood. In a 12-year study, whole blood and tissue samples were collected from 212 pigs across 19 districts in Uganda. Polymerase chain reaction (PCR) assays were used to determine ASFV infection status and genotyping was completed using a commercial porcine array. The point prevalence of ASF was calculated for each district, and breed composition origins were quantified for the sampled pigs by implementing established ancestry analyses. Genome-wide associated studies (GWAS) were conducted using all available domestic swine samples (full study population; n = 206) as well as a reduced dataset (farm-level study population; n = 129). This study revealed a greater number of ASFV-positive pigs in border districts than in non-border districts, a high level of admixture among domestic pigs sampled from Ugandan smallholder farms, and 48 loci that were associated with ASFV infection status. The discovery of 48 significant SNPs and 28 putative candidate genes may imply the possibility of heritability for resistance to ASFV. However, additional investigations in ASFV-endemic regions are required to fully elucidate the heritability of ASFV susceptibility among surviving pigs in Uganda.

Can demographic histories explain long-term isolation and recent pulses of asymmetric gene flow between highly divergent grey fox lineages?

Secondary contact zones between deeply divergent, yet interfertile, lineages provide windows into the speciation process. North American grey foxes (Urocyon cinereoargenteus) are divided into western and eastern lineages that diverged approximately 1 million years ago. These ancient lineages currently hybridize in a relatively narrow zone of contact in the southern Great Plains, a pattern more commonly observed in smaller-bodied taxa, which suggests relatively recent contact after a long period of allopatry. Based on local ancestry inference with whole-genome sequencing (n = 43), we identified two distinct Holocene pulses of admixture. The older pulse (500-3500 YBP) reflected unidirectional gene flow from east to west, whereas the more recent pulse (70-200 YBP) of admixture was bi-directional. Augmented with genotyping-by-sequencing data from 216 additional foxes, demographic analyses indicated that the eastern lineage declined precipitously after divergence, remaining small throughout most of the late Pleistocene, and expanding only during the Holocene. Genetic diversity in the eastern lineage was highest in the southeast and lowest near the contact zone, consistent with a westward expansion. Concordantly, distribution modelling indicated that during their isolation, the most suitable habitat occurred far east of today's contact zone or west of the Great Plains. Thus, long-term isolation was likely caused by the small, distant location of the eastern refugium, with recent contact reflecting a large increase in suitable habitat and corresponding demographic expansion from the eastern refugium. Ultimately, long-term isolation in grey foxes may reflect their specialized bio-climatic niche. This system presents an opportunity for future investigation of potential pre- and post-zygotic isolating mechanisms.

Genomic analyses of gray fox lineages suggest ancient divergence and secondary contact in the southern Great Plains.

The gray fox (Urocyon cinereoargenteus) lineage diverged from all other extant canids at their most basal node and is restricted to the Americas. Previous mitochondrial analysis from coastal populations identified deeply divergent (up to 1 Mya) eastern and western lineages that predate most intraspecific splits in carnivores. We conducted genotyping by sequencing and mitochondrial analysis on gray foxes sampled across North America to determine geographic concordance between nuclear and mitochondrial contact zones and divergence times. We also estimated the admixture within the contact zone between eastern and western gray foxes based on nuclear DNA. Both datasets confirmed that eastern and western lineages met in the southern Great Plains (i.e. Texas and Oklahoma), where they maintained high differentiation. Admixture was generally low, with the majority of admixed individuals carrying <10% ancestry from the other lineage. Divergence times confirmed a mid-Pleistocene split, similar to the mitochondrial estimates. Taken together, findings suggest gray fox lineages represent an ancient divergence event, far older than most intraspecific divergences in North American carnivores. Low admixture may reflect a relatively recent time since secondary contact (e.g. post-Pleistocene) or, alternatively, ecological or reproductive barriers between lineages. Though further research is needed to disentangle these factors, our genomic investigation suggests species-level divergence exists between eastern and western gray fox lineages.

Mixed ancestry from wild and domestic lineages contributes to the rapid expansion of invasive feral swine.

Invasive alien species are a significant threat to both economic and ecological systems. Identifying the processes that give rise to invasive populations is essential for implementing effective control strategies. We conducted an ancestry analysis of invasive feral swine (Sus scrofa, Linnaeus, 1758), a highly destructive ungulate that is widely distributed throughout the contiguous United States, to describe introduction pathways, sources of newly emergent populations and processes contributing to an ongoing invasion. Comparisons of high-density single nucleotide polymorphism genotypes for 6,566 invasive feral swine to a comprehensive reference set of S. scrofa revealed that the vast majority of feral swine were of mixed ancestry, with dominant genetic associations to Western heritage breeds of domestic pig and European populations of wild boar. Further, the rapid expansion of invasive feral swine over the past 30 years was attributable to secondary introductions from established populations of admixed ancestry as opposed to direct introductions of domestic breeds or wild boar. Spatially widespread genetic associations of invasive feral swine to European wild boar deviated strongly from historical S. scrofa introduction pressure, which was largely restricted to domestic pigs with infrequent, localized wild boar releases. The deviation between historical introduction pressure and contemporary genetic ancestry suggests wild boar-hybridization may contribute to differential fitness in the environment and heightened invasive potential for individuals of admixed domestic pig-wild boar ancestry.

Invasive wild pig (Sus scrofa) diets on barrier islands in the southeastern United States.

BACKGROUND: Biological invasions are a leading cause of reductions in global biodiversity. Islands are particularly sensitive to invasions, which often result in cascading impacts throughout island communities. Wild pigs (Sus scrofa) are globally invasive and pose threats to numerous taxa and ecosystems, particularly for islands where they have contributed to declines of many endemic species. However, the impacts of wild pig diet on the flora and fauna remain understudied in many island systems. RESULTS: We used DNA metabarcoding of wild pig fecal samples to quantify the seasonal diet composition of wild pigs on three barrier islands in the southeastern United States. Wild pigs exhibited a diverse diet dominated by plants, but also including marine and terrestrial animals. The diet composition of plants varied seasonally and between islands. Consumption of invertebrates also changed seasonally, with a shift to coastal invertebrates, particularly crabs, in spring and summer. Vertebrates were found in <10% of samples, but spanned broad taxa including amphibians, fish, mammals, and reptiles. Species consumed by wild pigs indicate that wild pigs use a variety of habitats within barrier islands for foraging, including maritime forests, saltmarshes, and beaches. CONCLUSIONS: An observed shift to beach foraging during sea turtle nesting season suggests wild pigs have potential to hinder nesting success on islands without established management programs. These findings provide insight into the diverse diets of wild pigs on barrier islands and highlight the need for removal of wild pigs from sensitive island ecosystems because of their potential impacts to native plant and animal communities. © 2024 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Biodiversity indices and Random Forests reveal the potential for striped skunk (Mephitis mephitis) fecal microbial communities to function as a biomarker for oral rabies vaccination.

Wildlife disease surveillance and monitoring poses unique challenges when assessing rates of population vaccination, immunity, or infection prevalence. Non-invasively detected biomarkers can help reduce risk to both animal and field personnel during wildlife disease management activities. In this study, we investigated the utility of fecal microbiome data collected from captive striped skunks (Mephitis mephitis) in predicting rabies virus vaccination and infection status. We sequenced the hypervariable region 4 (V4) of the bacterial 16S gene and estimated alpha and beta diversity across timepoints in three groups of skunks: vaccination then rabies virus infection, sham vaccination then rabies virus infection, and rabies virus infected without vaccination. Alpha diversity did not differ among treatment groups but beta diversity between treatments was statistically significant. The phyla Firmicutes and Proteobacteria were dominant among all samples. Using Random Forests, we identified operational taxonomic units (OTUs) that greatly influenced classification of fecal samples into treatment groups. Each of these OTUs was correlated with fecal volatile organic compounds detected from the samples for companion treatment groups in another study. This research is the first to highlight striped skunk microbiome biodiversity as a vaccination biomarker which pushes the frontier on alternative methods for surveillance and monitoring of vaccination and disease in wildlife populations.

DNA metabarcoding reveals consumption of diverse community of amphibians by invasive wild pigs (Sus scrofa) in the southeastern United States.

Invasive wild pigs (Sus scrofa) are one of the most widespread, destructive vertebrate species globally. Their success can largely be attributed to their generalist diets, which are dominated by plant material but also include diverse animal taxa. Wild pigs are demonstrated nest predators of ground-nesting birds and reptiles, and likely pose a threat to amphibians given their extensive overlap in wetland use. DNA metabarcoding of fecal samples from 222 adult wild pigs culled monthly from 2017 to 2018 revealed a diverse diet dominated by plant material, with 166 plant genera from 56 families and 18 vertebrate species identified. Diet composition varied seasonally with availability for plants and was consistent between sexes. Amphibians were the most frequent vertebrate group consumed and represented the majority of vertebrate species detected, suggesting amphibians are potentially vulnerable to predation by wild pigs in our study region. Mammal, reptile, and bird species were also detected in pig diets, but infrequently. Our results highlight the need for research on the impacts of wild pigs on amphibians to better inform management and conservation of imperiled species.

Genomic regions associated with pseudorabies virus infection status in naturally infected feral swine (Sus scrofa).

Pseudorabies virus (PRV)-the causative agent of Aujeszky's disease-was eliminated from commercial pig production herds in the United States (US) in 2004; however, PRV remains endemic among invasive feral swine (Sus scrofa). The circulation of PRV among abundant, widespread feral swine populations poses a sustained risk for disease spillover to production herds. Risk-based surveillance has been successfully implemented for PRV in feral swine populations in the US. However, understanding the role of host genetics in infection status may offer new insights into the epidemiology and disease dynamics of PRV that can be applied to management strategies. Genetic mechanisms underlying host susceptibility to PRV are relatively unknown; therefore, we sought to identify genomic regions associated with PRV infection status among naturally infected feral swine using genome-wide association studies (GWAS) and gene set enrichment analysis of single nucleotide polymorphism data (GSEA-SNP). Paired serological and genotypic data were collected from 6,081 feral swine distributed across the invaded range within the contiguous US. Three complementary study populations were developed for GWAS: 1) comprehensive population consisting of feral swine throughout the invaded range within the contiguous US; 2) population of feral swine under high, but temporally variable PRV infection pressure; and 3) population of feral swine under temporally stable, high PRV infection pressure. We identified one intronic SNP associated with PRV infection status within candidate gene AKAP6 on autosome 7. Various gene sets linked to metabolic pathways were enriched in the GSEA-SNP. Ultimately, improving disease surveillance efforts in feral swine will be critical to further understanding of the role host genetics play in PRV infection status, helping secure the health of commercial pork production.

Are rabid raccoons (Procyon lotor) ready for the rapture? Determining the geographic origin of rabies virus-infected raccoons using RADcapture and microhaplotypes.

North America is recognized for the exceptional richness of rabies virus (RV) wildlife reservoir species. Management of RV is accomplished through vaccination targeting mesocarnivore reservoir populations, such as the raccoon (Procyon lotor) in Eastern North America. Raccoons are a common generalist species, and populations may reach high densities in developed areas, which can result in contact with humans and pets with potential exposures to the raccoon variant of RV throughout the eastern United States. Understanding the spatial movement of RV by raccoon populations is important for monitoring and refining strategies supporting the landscape-level control and local elimination of this lethal zoonosis. We developed a high-throughput genotyping panel for raccoons based on hundreds of microhaplotypes to identify population structure and genetic diversity relevant to rabies management programs. Throughout the eastern United States, we identified hierarchical population genetic structure with clusters that were connected through isolation-by-distance. We also illustrate that this genotyping approach can be used to support real-time management priorities by identifying the geographic origin of a rabid raccoon that was collected in an area of the United States that had been raccoon RV-free for 8 years. The results from this study and the utility of the microhaplotype panel and genotyping method will provide managers with information on raccoon ecology that can be incorporated into future management decisions.

Evaluating noninvasive methods for estimating cestode prevalence in a wild carnivore population.

Helminth infections are cryptic and can be difficult to study in wildlife species. Helminth research in wildlife hosts has historically required invasive animal handling and necropsy, while results from noninvasive parasite research, like scat analysis, may not be possible at the helminth species or individual host levels. To increase the utility of noninvasive sampling, individual hosts can be identified by applying molecular methods. This allows for longitudinal sampling of known hosts and can be paired with individual-level covariates. Here we evaluate a combination of methods and existing long-term monitoring data to identify patterns of cestode infections in gray wolves in Yellowstone National Park. Our goals were: (1) Identify the species and apparent prevalence of cestodes infecting Yellowstone wolves; (2) Assess the relationships between wolf biological and social characteristics and cestode infections; (3) Examine how wolf samples were affected by environmental conditions with respect to the success of individual genotyping. We collected over 200 wolf scats from 2018-2020 and conducted laboratory analyses including individual wolf genotyping, sex identification, cestode identification, and fecal glucocorticoid measurements. Wolf genotyping success rate was 45%, which was higher in the winter but decreased with higher precipitation and as more time elapsed between scat deposit and collection. One cestode species was detected in 28% of all fecal samples, and 38% of known individuals. The most common infection was Echinococcus granulosus sensu lato (primarily E. canadensis). Adult wolves had 4x greater odds of having a cestode infection than pups, as well as wolves sampled in the winter. Our methods provide an alternative approach to estimate cestode prevalence and to linking parasites to known individuals in a wild host system, but may be most useful when employed in existing study systems and when field collections are designed to minimize the time between fecal deposition and collection.

Sex and nest type influence avian blood parasite prevalence in a high-elevation bird community.

BACKGROUND: The prevalence of avian haemosporidian parasites and the factors influencing infection in the Colorado Rocky Mountains are largely unknown. With climate change expected to promote the expansion of vector and avian blood parasite distributions, baseline knowledge and continued monitoring of the prevalence and diversity of these parasites is needed. METHODS: Using an occupancy modeling framework, we conducted a survey of haemosporidian parasite species infecting an avian community in the Colorado Rocky Mountains in order to estimate the prevalence and diversity of blood parasites and to investigate species-level and individual-level characteristics that may influence infection. RESULTS: We estimated the prevalence and diversity of avian Haemosporidia across 24 bird species, detecting 39 parasite haplotypes. We found that open-cup nesters have higher Haemoproteus prevalence than cavity or ground nesters. Additionally, we found that male Ruby-crowned Kinglets, White-crowned Sparrows, and Wilson's Warblers have higher Haemoproteus prevalence compared to other host species. Plasmodium prevalence was relatively low (5%), consistent with the idea that competent vectors may be rare at high altitudes. CONCLUSIONS: Our study presents baseline knowledge of haemosporidian parasite presence, prevalence, and diversity among avian species in the Colorado Rocky Mountains and adds to our knowledge of host-parasite relationships of blood parasites and their avian hosts.

Temporal and Spatial Blood Feeding Patterns of Urban Mosquitoes in the San Juan Metropolitan Area, Puerto Rico.

Urban ecosystems are a patchwork of habitats that host a broad diversity of animal species. Insects comprise a large portion of urban biodiversity which includes many pest species, including those that transmit pathogens. Mosquitoes (Diptera: Culicidae) inhabit urban environments and rely on sympatric vertebrate species to complete their life cycles, and in this process transmit pathogens to animals and humans. Given that mosquitoes feed upon vertebrates, they can also act as efficient samplers that facilitate detection of vertebrate species that utilize urban ecosystems. In this study, we analyzed DNA extracted from mosquito blood meals collected temporally in multiple neighborhoods of the San Juan Metropolitan Area, Puerto Rico to evaluate the presence of vertebrate fauna. DNA was collected from 604 individual mosquitoes that represented two common urban species, Culex quinquefasciatus (n = 586) and Aedes aegypti (n = 18). Culex quinquefasciatus fed on 17 avian taxa (81.2% of blood meals), seven mammalian taxa (17.9%), and one reptilian taxon (0.85%). Domestic chickens dominated these blood meals both temporally and spatially, and no statistically significant shift from birds to mammals was detected. Aedes aegypti blood meals were from a less diverse group, with two avian taxa (11.1%) and three mammalian taxa (88.9%) identified. The blood meals we identified provided a snapshot of the vertebrate community in the San Juan Metropolitan Area and have potential implications for vector-borne pathogen transmission.

Population genomic transformations induced by isolation of wild bird avian influenza viruses (Orthomyxoviridae) in embryonated chicken eggs.

Isolation and cultivation of wild-type viruses in model organism cells or tissues is standard practice in virology. Oftentimes, the virus host species is distantly related to the species from which the culture system was developed. Thus, virus culture in these tissues and cells basically constitutes a host jump, which can lead to genomic changes through genetic drift and/or adaptation to the culture system. We directly sequenced 70 avian influenza virus (Orthomyxoviridae) genomes from oropharyngeal/cloacal swabs collected from wild bird species and paired virus isolates propagated from the same samples following isolation in specific-pathogen-free embryonated chicken eggs. The data were analyzed using population genetic approaches including evaluation of single nucleotide polymorphism (SNP) frequencies and divergence with pooled-sequencing analyses, consensus sequence placement in neighbor-joining trees, and haplotype reconstruction and networks. We found that propagation of virus in eggs leads to skewed SNP mutation spectra with some SNPs going to fixation. Both synonymous and nonsynonmous SNP frequencies shifted. We found multiple consensus sequences that differed between the swabs and the isolates, with some sequences from the same sample falling into divergent genetic clusters. Twenty of 23 coinfections detected had different dominant subtypes following virus isolation, thus sequences from both the swab and isolate were needed to obtain full subtype data. Haplotype networks revealed haplotype frequency shifts and the appearance or loss of low-frequency haplotypes following isolation. The results from this study revealed that isolation of wild bird avian influenza viruses in chicken eggs leads to skewed populations that are different than the input populations. Consensus sequence changes from virus isolation can lead to flawed phylogenetic inferences, and subtype detection is biased. These results suggest that for genomic studies of wild bird influenza viruses the biological field should move away from chicken egg isolation towards directly sequencing the virus from host samples.

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.

Strategic considerations for invasive species managers in the utilization of environmental DNA (eDNA): steps for incorporating this powerful surveillance tool.

Invasive species surveillance programs can utilize environmental DNA sampling and analysis to provide information on the presence of invasive species. Wider utilization of eDNA techniques for invasive species surveillance may be warranted. This paper covers topics directed towards invasive species managers and eDNA practitioners working at the intersection of eDNA techniques and invasive species surveillance. It provides background information on the utility of eDNA for invasive species management and points to various examples of its use across federal and international programs. It provides information on 1) why an invasive species manager should consider using eDNA, 2) deciding if eDNA can help with the manager's surveillance needs, 3) important components to operational implementation, and 4) a high-level overview of the technical steps necessary for eDNA analysis. The goal of this paper is to assist invasive species managers in deciding if, when, and how to use eDNA for surveillance. If eDNA use is elected, the paper provides guidance on steps to ensure a clear understanding of the strengths and limitation of the methods and how results can be best utilized in the context of invasive species surveillance.

Predicting whole genome sequencing success for archived avian influenza virus (Orthomyxoviridae) samples using real-time and droplet PCRs.

Long-term viral archives are valuable sources of research data. Each archive can store hundreds of thousands of diverse sample types. In the current era of whole genome sequencing, archived samples become a rich source of evolutionary and epidemiological data that can span years, and even decades. However, the ability to obtain high quality viral whole genome sequences from samples of various types, age, and quality is inconsistent. A minimum quality threshold that helps predict the best success of obtaining high quality genomic sequences for both recent and archived samples is highly valuable. Real-time reverse transcription PCR (rrt-PCR) and droplet digital PCR (ddPCR) are useful tools to evaluate nucleic acid integrity. We hypothesized that diagnostic rrt-PCR and ddPCR data for avian influenza virus (AIV) can predict viral whole genome sequencing success. To test this hypothesis we used RNA extracted from cloacal and oropharyngeal swabs stored in the USDA-APHIS National Wildlife Disease Program Wildlife Tissue Archive. We determined that a specific rrt-PCR Cq value or ddPCR copies/µL resulted in recovery of complete sequences of all eight AIV gene segments. We used logistic regression to estimate probabilities of whole genome recovery at 0.95 (Cq = 15, copies/µL = 49,350), 0.75 (Cq = 24, copies/µL = 16,800), 0.50 (Cq = 29, copies/µL = <1), and 0.25 (Cq = 235, copies/µL = <1). We also identified values at which we predictably recovered HA and NA segments for diagnosing subtypes (Cq = 27.29; copies/µL = 757.50). This approach will allow researchers to assess the potential success of AIV whole genome recovery from diagnostic samples collected in routine AIV surveillance.

Technical Limitations Associated With Molecular Barcoding of Arthropod Bloodmeals Taken From North American Deer Species.

Accurate species-level identification of the source of arthropod bloodmeals is important for deciphering blood feeding patterns of field-collected specimens. Cytochrome c oxidase I (COI) mitochondrial gene sequencing has been used for this purpose; however, species resolution can be difficult to obtain from certain vertebrate genera, including Odocoileus. Sanger sequencing of mitochondrial genes was employed to identify the bloodmeal source of wild-caught mosquitoes trapped in Greeley, Colorado. Initial sequencing of the COI gene of mitochondrial DNA in bloodmeals was inadequate for species-level resolution of bloodmeals from deer in the genus Odocoileus, with current databases returning low fidelity matches to multiple genera. The use of the hypervariable D loop of the control region provided species-level identification of white-tailed deer (Order: Artiodactyla, Family: Cervidae, Odocoileus virginianus); however, taxonomic identification was successful only to genus for mule (O. hemionus hemionus) and black-tailed deer (O. hemionus columbianus). We advocate the use of multiple loci for bloodmeal analysis and the buildout of available databases to include multiple mitochondrial reference genes for reliable host species identification.