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1.
Artigo em Inglês | MEDLINE | ID: mdl-38775097

RESUMO

Background: Throughout the Americas, Lyssavirus rabies (RV) perpetuates as multiple variants among bat and mesocarnivore species. Interspecific RV spillover occurs on occasion, but clusters and viral host shifts are rare. The spillover and host shift of a big brown bat (Eptesicus fuscus) RV variant Ef-W1 into mesocarnivores was reported previously on several occasions during 2001-2009 in Flagstaff, Arizona, USA, and controlled through rabies vaccination of target wildlife. During autumn 2021, a new cluster of Ef-W1 RV cases infecting striped skunks (Mephitis mephitis) was detected from United States Department of Agriculture enhanced rabies surveillance in Flagstaff. The number of Ef-W1 RV spillover cases within a short timeframe suggested the potential for transmission between skunks and an emerging host shift. Materials and Methods: Whole and partial RV genomic sequencing was performed to evaluate the phylogenetic relationships of the 2021-2023 Ef-W1 cases infecting striped skunks with earlier outbreaks. Additionally, real-time reverse-transcriptase PCR (rtRT-PCR) was used to opportunistically compare viral RNA loads in brain and salivary gland tissues of naturally infected skunks. Results: Genomic RV sequencing revealed that the origin of the 2021-2023 epizootic of Ef-W1 RV was distinct from the multiple outbreaks detected from 2001-2009. Naturally infected skunks with the Ef-W1 RV showed greater viral RNA loads in the brain, but equivalent viral RNA loads in the mandibular salivary glands, compared to an opportunistic sample of skunks naturally infected with a South-Central skunk RV from northern Colorado, USA. Conclusion: Considering a high risk for onward transmission and spread of the Ef-W1 RV in Flagstaff, public outreach, enhanced rabies surveillance, and control efforts, focused on education, sample characterization, and vaccination, have been ongoing since 2021 to mitigate and prevent the spread and establishment of Ef-W1 RV in mesocarnivores.

2.
J Wildl Dis ; 60(3): 745-752, 2024 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-38685759

RESUMO

Wildlife translocation and cross-species transmission can impede control and elimination of emerging zoonotic diseases. Tracking the geographic origin of both host and virus (i.e., translocation versus local infection) may help determine the most effective response when high-risk cases of emerging pathogens are identified in wildlife. In May 2022, a coyote (Canis latrans) infected with the raccoon (Procyon lotor) rabies virus variant (RRV) was collected in Lewis County, West Virginia, USA, an area free from RRV. We applied host population genomics and RRV phylogenetic analyses to determine the most likely geographic origin of the rabid coyote. Coyote genomic analyses included animals from multiple eastern states bordering West Virginia, with the probable origin of the rabid coyote being the county of collection. The RRV phylogenetic analyses included cases detected from West Virginia and neighboring states, with most similar RRV sequences collected in a county 80 km to the northeast, within the oral rabies vaccination zone. The combined results suggest that the coyote was infected in an RRV management area and carried the RRV to Lewis County, a pattern consistent with coyote local movement ecology. Distant cross-species transmission and subsequent host movement presents a low risk for onward transmission in raccoon populations. This information helped with emergency response decision-making, thereby saving time and resources.


Assuntos
Coiotes , Filogenia , Vírus da Raiva , Raiva , Animais , Coiotes/virologia , West Virginia/epidemiologia , Raiva/veterinária , Raiva/epidemiologia , Vírus da Raiva/genética , Vírus da Raiva/isolamento & purificação , Vírus da Raiva/classificação , Guaxinins/virologia , Animais Selvagens
3.
Evol Appl ; 16(12): 1937-1955, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-38143904

RESUMO

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.

4.
PLoS One ; 18(8): e0285852, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37607164

RESUMO

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.


Assuntos
Microbiota , Vírus da Raiva , Raiva , Animais , Mephitidae , Algoritmo Florestas Aleatórias , Animais Selvagens , Biodiversidade , Biomarcadores , Complicações Pós-Operatórias
5.
Insects ; 12(2)2021 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-33540671

RESUMO

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.

6.
Infect Genet Evol ; 90: 104505, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-32827730

RESUMO

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.


Assuntos
Galinhas , Genoma , Vírus da Influenza A/fisiologia , Influenza Aviária/virologia , Óvulo/virologia , Polimorfismo de Nucleotídeo Único , Animais , Embrião de Galinha , Galinhas/genética , Cloaca/virologia , Orofaringe/virologia
8.
J Med Entomol ; 57(6): 2002-2006, 2020 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-32574357

RESUMO

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.


Assuntos
Culicidae/fisiologia , Código de Barras de DNA Taxonômico/instrumentação , Cervos/fisiologia , Cadeia Alimentar , Animais , Colorado , Dieta , Complexo IV da Cadeia de Transporte de Elétrons/análise , Comportamento Alimentar , Controle de Mosquitos/instrumentação
9.
Sci Rep ; 10(1): 8093, 2020 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-32415136

RESUMO

Antimicrobial use in livestock production is a driver for the development and proliferation of antimicrobial resistance (AMR). Wildlife interactions with livestock, acquiring associated AMR bacteria and genes, and wildlife's subsequent dispersal across the landscape are hypothesized to play an important role in the ecology of AMR. Here, we examined priority AMR phenotypes and genotypes of Escherichia coli isolated from the gastrointestinal tracts of European starlings (Sturnus vulgaris) found on concentrated animal feeding operations (CAFOs). European starlings may be present in high numbers on CAFOs (>100,000 birds), interact with urban environments, and can migrate distances exceeding 1,500 km in North America. In this study, 1,477 European starlings from 31 feedlots in five U.S. states were sampled for E. coli resistant to third generation cephalosporins (3G-C) and fluoroquinolones. The prevalence of 3G-C and fluoroquinolone-resistant E. coli was 4% and 10%, respectively. Multidrug resistance in the E. coli isolates collected (n = 236) was common, with the majority of isolates displaying resistance to six or more classes of antibiotics. Genetic analyses of a subset of these isolates identified 94 genes putatively contributing to AMR, including seven class A and C ß-lactamases as well as mutations in gyrA and parC recognized to confer resistance to quinolones. Phylogenetic and subtyping assessments showed that highly similar isolates (≥99.4% shared core genome, ≥99.6% shared coding sequence) with priority AMR were found in birds on feedlots separated by distances exceeding 150 km, suggesting that European starlings could be involved in the interstate dissemination of priority AMR bacteria.


Assuntos
Ração Animal/análise , Antibacterianos/farmacologia , Doenças das Aves/tratamento farmacológico , Farmacorresistência Bacteriana Múltipla , Infecções por Escherichia coli/veterinária , Escherichia coli/efeitos dos fármacos , Trato Gastrointestinal/efeitos dos fármacos , Estorninhos/microbiologia , Animais , Doenças das Aves/epidemiologia , Doenças das Aves/microbiologia , Escherichia coli/isolamento & purificação , Infecções por Escherichia coli/tratamento farmacológico , Infecções por Escherichia coli/epidemiologia , Infecções por Escherichia coli/microbiologia , Europa (Continente)/epidemiologia , Trato Gastrointestinal/microbiologia , Filogenia
10.
J Virol Methods ; 276: 113777, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31730870

RESUMO

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.


Assuntos
Aves/virologia , Vírus da Influenza A/genética , Influenza Aviária/virologia , Reação em Cadeia da Polimerase/métodos , Sequenciamento Completo do Genoma , Animais , Animais Selvagens/virologia , Genoma Viral , Vírus da Influenza A/classificação , RNA Viral/genética , Análise de Regressão
11.
Philos Trans R Soc Lond B Biol Sci ; 374(1782): 20180346, 2019 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-31401963

RESUMO

Reassortment is an evolutionary mechanism by which influenza A viruses (IAV) generate genetic novelty. Reassortment is an important driver of host jumps and is widespread according to retrospective surveillance studies. However, predicting the epidemiological risk of reassortant emergence in novel hosts from surveillance data remains challenging. IAV strains persist and co-occur in the environment, promoting co-infection during environmental transmission. These conditions offer opportunity to understand reassortant emergence in reservoir and spillover hosts. Specifically, environmental RNA could provide rich information for understanding the evolutionary ecology of segmented viruses, and transform our ability to quantify epidemiological risk to spillover hosts. However, significant challenges with recovering and interpreting genomic RNA from the environment have impeded progress towards predicting reassortant emergence from environmental surveillance data. We discuss how the fields of genomics, experimental ecology and epidemiological modelling are well positioned to address these challenges. Coupling quantitative disease models and natural transmission studies with new molecular technologies, such as deep-mutational scanning and single-virus sequencing of environmental samples, should dramatically improve our understanding of viral co-occurrence and reassortment. We define observable risk metrics for emerging molecular technologies and propose a conceptual research framework for improving accuracy and efficiency of risk prediction. This article is part of the theme issue 'Dynamic and integrative approaches to understanding pathogen spillover'.


Assuntos
Monitoramento Epidemiológico/veterinária , Influenza Humana/transmissão , Infecções por Orthomyxoviridae/veterinária , Animais , Animais Selvagens , Humanos , Vírus da Influenza A/fisiologia , Infecções por Orthomyxoviridae/transmissão , Medição de Risco/métodos
12.
PLoS Pathog ; 14(12): e1007417, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30507946

RESUMO

Genetic reassortment between influenza A viruses (IAVs) facilitate emergence of pandemic strains, and swine are proposed as a "mixing vessel" for generating reassortants of avian and mammalian IAVs that could be of risk to mammals, including humans. However, how a transmissible reassortant emerges in swine are not well understood. Genomic analyses of 571 isolates recovered from nasal wash samples and respiratory tract tissues of a group of co-housed pigs (influenza-seronegative, avian H1N1 IAV-infected, and swine H3N2 IAV-infected pigs) identified 30 distinct genotypes of reassortants. Viruses recovered from lower respiratory tract tissues had the largest genomic diversity, and those recovered from turbinates and nasal wash fluids had the least. Reassortants from lower respiratory tracts had the largest variations in growth kinetics in respiratory tract epithelial cells, and the cold temperature in swine nasal cells seemed to select the type of reassortant viruses shed by the pigs. One reassortant in nasal wash samples was consistently identified in upper, middle, and lower respiratory tract tissues, and it was confirmed to be transmitted efficiently between pigs. Study findings suggest that, during mixed infections of avian and swine IAVs, genetic reassortments are likely to occur in the lower respiratory track, and tissue tropism is an important factor selecting for a transmissible reassortant.


Assuntos
Vírus da Influenza A Subtipo H1N1 , Vírus da Influenza A Subtipo H3N2 , Infecções por Orthomyxoviridae , Recombinação Genética/genética , Tropismo Viral , Animais , Coinfecção , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/patogenicidade , Vírus da Influenza A Subtipo H3N2/genética , Vírus da Influenza A Subtipo H3N2/patogenicidade , Infecções por Orthomyxoviridae/genética , Infecções por Orthomyxoviridae/transmissão , Vírus Reordenados/genética , Vírus Reordenados/patogenicidade , Infecções Respiratórias/virologia , Suínos
13.
Viruses ; 9(11)2017 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-29117106

RESUMO

Since 2000, scientists and students from the greater Rocky Mountain region, along with invited speakers, both national and international, have gathered at the Mountain Campus of Colorado State University to discuss their area of study, present recent findings, establish or strengthen collaborations, and mentor the next generation of virologists and prionologists through formal presentations and informal discussions concerning science, grantsmanship and network development. This year, approximately 100 people attended the 17th annual Rocky Mountain Virology Association meeting, that began with a keynote presentation, and featured 29 oral and 35 poster presentations covering RNA and DNA viruses, prions, virus-host interactions and guides to successful mentorship. Since the keynote address focused on the structure and function of Zika and related flaviviruses, a special session was held to discuss RNA control. The secluded meeting at the foot of the Colorado Rocky Mountains gave ample time for in-depth discussions amid the peak of fall colors in the aspen groves while the random bear provided excitement. On behalf of the Rocky Mountain Virology Association, this report summarizes the >50 reports.


Assuntos
Vírus de DNA , Príons , Vírus de RNA , Viroses , Dengue/transmissão , Humanos , Viroses/virologia , Zika virus/metabolismo , Infecção por Zika virus/transmissão , Infecção por Zika virus/virologia
14.
Parasit Vectors ; 10(1): 169, 2017 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-28376843

RESUMO

BACKGROUND: Biting midges in the genus Culicoides (Diptera; Ceratopogonidae) have been implicated in the transmission of a number of parasites and highly pathogenic viruses. In North America, the complete transmission cycles of many of these pathogens need further elucidation. One way to increase our knowledge about the evolution and ecology of Culicoides species and the pathogens they transmit is to document the diversity of vertebrate hosts that Culicoides feed upon. Our objective was to identify the diversity of Culicoides hosts in the United States. RESULTS: We sequenced two vertebrate mitochondrial genes (cytochrome c oxidase subunit 1 and cytochrome b) from blood-engorged Culicoides to identify Culicoides species and their blood meals. We detected the mitochondrial DNA of 12 host species from seven different Culicoides species from three states. The majority of the identified blood meals were from the C. variipennis species complex in California. The hosts included both mammals and birds. We documented new host records for some of the Culicoides species collected. The majority of the mammalian hosts were large ungulate species but we also detected a lagomorph and a carnivore. The bird species that were detected included house finch and emu; the latter is evidence that the species in the C. variipennis species complex are not strictly mammalophilic. CONCLUSIONS: These results demonstrate that Culicoides will feed on multiple classes of vertebrates and may be more opportunistic in regards to host choice than previously thought. This knowledge can help with identification of susceptible host species, pathogen reservoirs, and new vector species which, in turn, will improve disease outbreak risk assessments.


Assuntos
Ceratopogonidae/fisiologia , Comportamento Alimentar , Especificidade de Hospedeiro , Animais , Sangue , Impressões Digitais de DNA , DNA Mitocondrial/química , DNA Mitocondrial/genética , Refeições , Análise de Sequência de DNA , Estados Unidos
15.
BMC Res Notes ; 9: 14, 2016 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-26738484

RESUMO

BACKGROUND: The greater sage-grouse (Centrocercus urophasianus) is a ground-nesting bird from the Northern Rocky Mountains and a species at risk of extinction in in multiple U.S. states and Canada. Herein we report results from a proof of concept that mitochondrial and nuclear DNAs from mammalian predator saliva could be non-invasively collected from depredated greater sage-grouse eggshells and carcasses and used for predator species identification. Molecular forensic approaches have been applied to identify predators from depredated remains as one strategy to better understand predator-prey dynamics and guide management strategies. This can aid conservation efforts by correctly identifying predators most likely to impact threatened and endangered species. DNA isolated from non-invasive samples around nesting sites (e.g. fecal or hair samples) is one method that can increase the success and accuracy of predator species identification when compared to relying on nest remains alone. RESULTS: Predator saliva DNA was collected from depredated eggshells and carcasses using swabs. We sequenced two partial fragments of two mitochondrial genes and obtained microsatellite genotypes using canid specific primers for species and individual identification, respectively. Using this multilocus approach we were able to identify predators, at least down to family, from 11 out of 14 nests (79%) and three out of seven carcasses (47%). Predators detected most frequently were canids (86%), while other taxa included rodents, a striped skunk, and cattle. We attempted to match the genotypes of individual coyotes obtained from eggshells and carcasses with those obtained from fecal samples and coyotes collected in the areas, but no genotype matches were found. CONCLUSION: Predation is a main cause of nest failure in ground-nesting birds and can impact reproduction and recruitment. To inform predator management for ground-nesting bird conservation, accurate identification of predator species is necessary. Considering predation can have a high impact on recruitment, predation events are very difficult to observe, and predator species are difficult to identify visually from nest remains, molecular approaches that reduce the need to observe or handle animals offer an additional tool to better understand predator-prey dynamics at nesting sites.


Assuntos
Aves/genética , Conservação dos Recursos Naturais , DNA/análise , Ciências Forenses/métodos , Mamíferos/fisiologia , Comportamento de Nidação , Comportamento Predatório , Animais , DNA Mitocondrial/genética , Genótipo , Repetições de Microssatélites/genética
16.
Mol Ecol Resour ; 14(2): 374-80, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24119154

RESUMO

Recent studies have demonstrated that detection of environmental DNA (eDNA) from aquatic vertebrates in water bodies is possible. The Burmese python, Python bivittatus, is a semi-aquatic, invasive species in Florida where its elusive nature and cryptic coloration make its detection difficult. Our goal was to develop a diagnostic PCR to detect P. bivittatus from water-borne eDNA, which could assist managers in monitoring this invasive species. First, we used captive P. bivittatus to determine whether reptilian DNA could be isolated and amplified from water samples. We also evaluated the efficacy of two DNA isolation methods and two DNA extraction kits commonly used in eDNA preparation. A fragment of the mitochondrial cytochrome b gene from P. bivittatus was detected in all water samples isolated with the sodium acetate precipitate and the QIAamp DNA Micro Kit. Next, we designed P. bivittatus-specific primers and assessed the degradation rate of eDNA in water. Our primers did not amplify DNA from closely related species, and we found that P. bivittatus DNA was consistently detectable up to 96 h. Finally, we sampled water from six field sites in south Florida. Samples from five sites, where P. bivittatus has been observed, tested positive for eDNA. The final site was negative and had no prior documented evidence of P. bivittatus. This study shows P. bivittatus eDNA can be isolated from water samples; thus, this method is a new and promising technique for the management of invasive reptiles.


Assuntos
Boidae/classificação , Boidae/crescimento & desenvolvimento , DNA/isolamento & purificação , Espécies Introduzidas , Reação em Cadeia da Polimerase/métodos , Animais , Boidae/genética , Citocromos b/genética , DNA/genética , Primers do DNA/genética , DNA Mitocondrial/genética , Florida , Água/química
17.
Mol Ecol ; 22(4): 956-71, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23279045

RESUMO

Global climate change is apparent within the Arctic and the south-western deserts of North America, with record drought in the latter reflected within 640,000 km(2) of the Colorado River Basin. To discern the manner by which natural and anthropogenic drivers have compressed Basin-wide fish biodiversity, and to establish a baseline for future climate effects, the Stream Hierarchy Model (SHM) was employed to juxtapose fluvial topography against molecular diversities of 1092 Bluehead Sucker (Catostomus discobolus). MtDNA revealed three geomorphically defined evolutionarily significant units (ESUs): Bonneville Basin, upper Little Colorado River and the remaining Colorado River Basin. Microsatellite analyses (16 loci) reinforced distinctiveness of the Bonneville Basin and upper Little Colorado River, but subdivided the Colorado River Basin into seven management units (MUs). One represents a cline of three admixed gene pools comprising the mainstem and its lower-gradient tributaries. Six others are not only distinct genetically but also demographically (i.e. migrants/generation <9.7%). Two of these (i.e. Grand Canyon and Canyon de Chelly) are defined by geomorphology, two others (i.e. Fremont-Muddy and San Raphael rivers) are isolated by sharp declivities as they drop precipitously from the west slope into the mainstem Colorado/Green rivers, another represents an isolated impoundment (i.e. Ringdahl Reservoir), while the last corresponds to a recognized subspecies (i.e. Zuni River, NM). Historical legacies of endemic fishes (ESUs) and their evolutionary potential (MUs) are clearly represented in our data, yet their arbiter will be the unrelenting natural and anthropogenic water depletions that will precipitate yet another conservation conflict within this unique but arid region.


Assuntos
Cipriniformes/genética , Variação Genética , Rios , Animais , Teorema de Bayes , Biodiversidade , Conservação dos Recursos Naturais , DNA Mitocondrial/genética , Clima Desértico , Monitoramento Ambiental , Evolução Molecular , Fluxo Gênico , Genética Populacional , Repetições de Microssatélites , América do Norte , Filogenia , Dinâmica Populacional , Análise de Sequência de DNA
18.
J Wildl Dis ; 49(4): 1060-2, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24502742

RESUMO

A wild-caught desert cottontail rabbit (Sylvilagus audubonii) from Colorado was observed to have large, pedunculated, dark cutaneous lesions on its abdomen and cylindrical masses on its mouth. Morphologically, the masses were consistent with previous reports of virally induced papillomas. Subsequent DNA analysis indicated widespread infection with cottontail rabbit papillomavirus.


Assuntos
Papillomavirus de Coelho Cottontail/isolamento & purificação , Papiloma/veterinária , Infecções por Papillomavirus/veterinária , Animais , Colorado/epidemiologia , Masculino , Papiloma/epidemiologia , Papiloma/patologia , Papiloma/virologia , Infecções por Papillomavirus/epidemiologia , Infecções por Papillomavirus/patologia , Infecções por Papillomavirus/virologia , Coelhos
19.
Am J Respir Crit Care Med ; 169(6): 764-9, 2004 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-14701708

RESUMO

Severe pulmonary hypertension (PH) occurs in a primary or "unexplained" form and in a group of secondary forms associated with a number of diseases. Because the lung tissue from patients with severe PH demonstrates complex vascular lesions, which contain inflammatory cells, we wondered whether the lung tissue from patients with severe PH was "under oxidative stress." We used immunohistochemistry to localize nitrotyrosine and 8-hydroxy guanosine in the lung tissue sections from patients with primary and secondary PH. In some lung tissue extracts, the eicosanoid metabolites 5-oxo-eicosatetraenoic acid, leukotriene B4 5-hydroxyeicosatetraenoic acid (HETE), 12-HETE, and 15-HETE were measured using mass spectroscopy, and superoxide dismutase amount and activity were measured. Nitrotyrosine expression was ubiquitous in all PH lungs, and 5-oxo-eicosatetraenoic acid and HETE levels were elevated in the lungs of patients with severe PH but not in those lungs that were from the patients with severe PH treated chronically with prostacyclin. We conclude that indeed the lungs from patients with severe PH are under oxidative stress and that chronic prostacyclin infusion has an antiinflammatory effect on the lung tissue.


Assuntos
Guanosina/análogos & derivados , Hipertensão Pulmonar/metabolismo , Pulmão/metabolismo , Estresse Oxidativo/fisiologia , Tirosina/análogos & derivados , Ácidos Araquidônicos/metabolismo , Guanosina/metabolismo , Humanos , Hipertensão Pulmonar/patologia , Pulmão/patologia , Índice de Gravidade de Doença , Superóxido Dismutase/metabolismo , Tirosina/metabolismo
20.
Biochem J ; 378(Pt 3): 919-28, 2004 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-14670077

RESUMO

Activation of cellular kinases and transcription factors mediates the early phase of the cellular response to chemically or biologically induced stress. In the present study we investigated the oxidant/antioxidant balance in Huh-7 cells expressing the HCV (hepatitis C virus) subgenomic replicon, and observed a 5-fold increase in oxidative stress during HCV replication. We used MnSOD (manganese-superoxide dismutase) as an indicator of the cellular antioxidant response, and found that its activity, protein levels and promoter activity were significantly increased, whereas Cu/ZnSOD was not affected. The oxidative stress-induced protein kinases p38 MAPK (mitogen-activated protein kinase) and JNK (c-Jun N-terminal kinase) were activated in the HCV repliconcontaining cells and in Huh-7 cells transduced with Ad-NS5A [a recombinant adenovirus encoding NS5A (non-structural protein 5A)], coupled with a 4-5-fold increase in AP-1 (activator protein-1) DNA binding. Ava.1 cells, which encode a replication-defective HCV replicon, showed no significant changes in MnSOD, p38 MAPK or JNK activity. The AP-1 inhibitors dithiothreitol and N -acetylcysteine, as well as a dominant negative AP-1 mutant, significantly reduced AP-1 activation, demonstrating that this activation is oxidative stress-related. Exogenous NS5A had no effect on AP-1 activation in vitro, suggesting that NS5A acts at the upstream targets of AP-1 involving p38 MAPK and JNK signalling cascades. AP-1-dependent gene expression was increased in HCV subgenomic replicon-expressing Huh-7 cells. MnSOD activation was blocked by inhibitors of JNK (JNKI1) and p38 MAPK (SB203580), but not by an ERK (extracellular-signal-regulated kinase) inhibitor (U0126), in HCV-replicating and Ad-NS5A-transduced cells. Our results demonstrate that cellular responses to oxidative stress in HCV subgenomic replicon-expressing and Ad-NS5A-transduced cells are regulated by two distinct signalling pathways involving p38 MAPK and JNK via AP-1 that is linked to increased oxidative stress and therefore to an increased antioxidant MnSOD response.


Assuntos
Hepacivirus/patogenicidade , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Superóxido Dismutase/metabolismo , Fator de Transcrição AP-1/fisiologia , Linhagem Celular , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Regulação Enzimológica da Expressão Gênica , Hepacivirus/genética , Hepacivirus/fisiologia , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Estresse Oxidativo , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-jun/biossíntese , Replicon , Superóxido Dismutase/genética , Ativação Transcricional , Proteínas não Estruturais Virais/metabolismo , Replicação Viral , Proteínas Quinases p38 Ativadas por Mitógeno
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