scholarly journals eDNA Increases the Detectability of Ranavirus Infection in an Alpine Amphibian Population

Viruses ◽  
2019 ◽  
Vol 11 (6) ◽  
pp. 526 ◽  
Author(s):  
Claude Miaud ◽  
Véronique Arnal ◽  
Marie Poulain ◽  
Alice Valentini ◽  
Tony Dejean
Keyword(s):  
Water Samples ◽  
Rana Temporaria ◽  
Clinical Signs ◽  
Environmental Dna ◽  
Diagnostic Techniques ◽  
Mitigation Measures ◽  
Activity Period ◽  
Amphibian Species ◽  
Tissue Samples ◽  
Species Activity

The early detection and identification of pathogenic microorganisms is essential in order to deploy appropriate mitigation measures. Viruses in the Iridoviridae family, such as those in the Ranavirus genus, can infect amphibian species without resulting in mortality or clinical signs, and they can also infect other hosts than amphibian species. Diagnostic techniques allowing the detection of the pathogen outside the period of host die-off would thus be of particular use. In this study, we tested a method using environmental DNA (eDNA) on a population of common frogs (Rana temporaria) known to be affected by a Ranavirus in the southern Alps in France. In six sampling sessions between June and September (the species’ activity period), we collected tissue samples from dead and live frogs (adults and tadpoles), as well as insects (aquatic and terrestrial), sediment, and water. At the beginning of the breeding season in June, one adult was found dead; at the end of July, a mass mortality of tadpoles was observed. The viral DNA was detected in both adults and tadpoles (dead or alive) and in water samples, but it was not detected in insects or sediment. In live frog specimens, the virus was detected from June to September and in water samples from August to September. Dead tadpoles that tested positive for Ranavirus were observed only on one date (at the end of July). Our results indicate that eDNA can be an effective alternative to tissue/specimen sampling and can detect Ranavirus presence outside die-offs. Another advantage is that the collection of water samples can be performed by most field technicians. This study confirms that the use of eDNA can increase the performance and accuracy of wildlife health status monitoring and thus contribute to more effective surveillance programs.

scholarly journals Long distance (>20 km) downstream detection of endangered stream frogs suggests an important role for eDNA in surveying for remnant amphibian populations

PeerJ ◽  
2021 ◽  
Vol 9 ◽  
pp. e12013
Author(s):  
Cecilia Villacorta-Rath ◽  
Conrad J. Hoskin ◽  
Jan M. Strugnell ◽  
Damien Burrows
Keyword(s):  
Water Samples ◽  
Survey Methods ◽  
Environmental Dna ◽  
Small Population ◽  
Population Declines ◽  
Long Distance ◽  
Amphibian Species ◽  
Filtration Method ◽  
North East ◽  
Water Volumes

Background Globally, amphibian species have suffered drastic population declines over the past 40 years. Hundreds of species are now listed as Critically Endangered, with many of these considered “possibly extinct”. Most of these species are stream-dwelling frogs inhabiting remote, montane areas, where remnant populations are hard to find using traditional surveys. Environmental DNA (eDNA) could revolutionize surveys for ‘missing’ and endangered amphibian populations by screening water samples from downstream sections to assess presence in the upstream catchments. However, the utility of this survey technique is dependent on quantifying downstream detection probability and distances. Methods Here we tested downstream detection distances in two endangered stream frogs (Litoria lorica and L. nannotis) that co-occur in a remote stream catchment in north-east Australia, and for which we know precise downstream distributional limits from traditional surveys. Importantly, the two last populations of L. lorica persist in this catchment: one small (~1,000 frogs) and one very small (~100 frogs). We conducted eDNA screening at a series of sites kilometers downstream from the populations using precipitation from two fixed water volumes (15 and 100 mL) and via water filtering (mean 1,480 L). Results We detected L. nannotis and the small L. lorica population (~1,000 frogs) at most sampling sites, including 22.8 km downstream. The filtration method was highly effective for far-downstream detection, as was precipitation from 100 mL water samples, which also resulted in consistent detections at the far-downstream sites (including to 22.8 km). In contrast, we had limited downstream detection success for the very small L. lorica population (~100 frogs). Discussion The ecological aspects of our study system, coupled with thorough traditional surveys, enabled us to measure downstream eDNA detection distances with accuracy. We demonstrate that eDNA from a small population of approximately 1,000 frogs can be detected as far as 22.8 km downstream from the population. Water filtration is considered best for eDNA detection of rare aquatic species—indeed it was effective in this study—but we also achieved far-downstream detections when precipitating eDNA from 100 mL water samples. Collecting small water volumes for subsequent precipitation in the lab is more practical than filtration when surveying remote areas. Our downstream detection distances (>20 km) suggest eDNA is a valuable tool for detecting rare stream amphibians. We provide recommendations on optimal survey methods.

scholarly journals Assessment of Environmental DNA for Detecting Presence of Imperiled Aquatic Amphibian Species in Isolated Wetlands

2015 ◽  
Vol 6 (2) ◽  
pp. 498-510 ◽  
Author(s):  
Anna M. McKee ◽  
Daniel L. Calhoun ◽  
William J. Barichivich ◽  
Stephen F. Spear ◽  
Caren S. Goldberg ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Water Samples ◽  
Survey Methods ◽  
Pinus Palustris ◽  
Environmental Dna ◽  
Breeding Habitat ◽  
Target Species ◽  
Amphibian Species ◽  
Ambystoma Bishopi ◽  
Ambystoma Cingulatum

Abstract Environmental DNA (eDNA) is an emerging tool that allows low-impact sampling for aquatic species by isolating DNA from water samples and screening for DNA sequences specific to species of interest. However, researchers have not tested this method in naturally acidic wetlands that provide breeding habitat for a number of imperiled species, including the frosted salamander (Ambystoma cingulatum), reticulated flatwoods salamanders (Ambystoma bishopi), striped newt (Notophthalmus perstriatus), and gopher frog (Lithobates capito). Our objectives for this study were to develop and optimize eDNA survey protocols and assays to complement and enhance capture-based survey methods for these amphibian species. We collected three or more water samples, dipnetted or trapped larval and adult amphibians, and conducted visual encounter surveys for egg masses for target species at 40 sites on 12 different longleaf pine (Pinus palustris) tracts. We used quantitative PCRs to screen eDNA from each site for target species presence. We detected flatwoods salamanders at three sites with eDNA but did not detect them during physical surveys. Based on the sample location we assumed these eDNA detections to indicate the presence of frosted flatwoods salamanders. We did not detect reticulated flatwoods salamanders. We detected striped newts with physical and eDNA surveys at two wetlands. We detected gopher frogs at 12 sites total, three with eDNA alone, two with physical surveys alone, and seven with physical and eDNA surveys. We detected our target species with eDNA at 9 of 11 sites where they were present as indicated from traditional surveys and at six sites where they were not detected with traditional surveys. It was, however, critical to use at least three water samples per site for eDNA. Our results demonstrate eDNA surveys can be a useful complement to traditional survey methods for detecting imperiled pond-breeding amphibians. Environmental DNA may be particularly useful in situations where detection probability using traditional survey methods is low or access by trained personnel is limited.

scholarly journals DNA-based biomonitoring in the tropics: Detection and control of Batrachochytrium dendrobatidis in Ecuadorian ecosystem

2021 ◽  
Vol 4 ◽  
Author(s):  
Lenin Riascos-Flores ◽  
Andrea Carrera ◽  
Leopoldo Naranjo ◽  
Jomira Yanez ◽  
Peter Goethals ◽  
...  
Keyword(s):  
Invasive Species ◽  
Water Samples ◽  
Large Scale ◽  
Batrachochytrium Dendrobatidis ◽  
Action Plan ◽  
Detection Methods ◽  
Population Declines ◽  
Amphibian Species ◽  
Tissue Samples ◽  
And Control

Batrachochytrium dendrobatidis (Bd) is a fungus that parasites vertebrates, and is associated with population declines worldwide in endemic amphibian species. As such, it is one of several invasive species which pose a serious threat to a variety of vertebrate hosts, in casu: amphibians. Detection of such invasive species is generally based on DNA-based methods where, for instance, swabs or tissue samples of candidate hosts are analysed for their presence. Any management strategy of these invasive species would greatly benefit from sensitive and rapid detection methods which can be applied at a large scale. The analysis of eDNA from the habitat of candidate host organisms may hold significant potential for this purpose. In this study, we compare the ability of eDNA from habitat samples with that of swab and/or tissue samples of candidate hosts to detect the presence of Bd in Ecuador. We collected individuals from the amphibians: Pristimantis (Anura: Craugastoridae), Rhinella (Anura: Bufonidae), Gastroteca (Anura: Hemiphractidae), from the endangered toad species of the genus Atelopus (Anura: Bufonidae) as well as water samples from different water bodies in Andean and coastal Ecuadorian areas. Samples were processed using a portable field molecular laboratory. Commercial primers for the internal transcribed spacer (ITS), in combination with a new set of primers designed from Bd sequences from tropical countries, were used. Positive PCR results from both types of samples were obtained within eight hours after sampling. Prevalence of BD was detected in eDNA, swab and tissue samples in four of the six ecosystems monitored -14 out of 26 water samples and 27 out of 43 amphibian of in total 12 species- including three endangered toad species (Atelopus balios, A. nanay, and the rediscovered A. bomolochos). Our results highlight the potential of eDNA-based monitoring to assess the presence and prevalence of Bd in Ecuadorian aquatic ecosystems, in accordance with the National Action Plan for the Conservation of Ecuadorian Amphibians. Furthermore, our field lab approach leads to reliable and fast results for the monitoring of invasive species in a tropical context of a pandemic.

scholarly journals Development and validation of a quantitative qPCR assay for detecting Natterjack toad (Epidalea calamita) eDNA samples

2021 ◽  
Author(s):  
Marina Reyne ◽  
Amanda M. Naaum ◽  
Ferdia Marnell ◽  
Neil Reid ◽  
Sarah J. Helyar
Keyword(s):  
Water Samples ◽  
Rapid Assessment ◽  
Pond Water ◽  
Qpcr Assay ◽  
Amphibian Species ◽  
Tissue Samples ◽  
Species Specific ◽  
Epidalea Calamita

AbstractThe Natterjack toad (Epidalea calamita) is the rarest amphibian species in Ireland, regionally Red-Listed as Endangered. We applied an eDNA approach to detect species presence in breeding pond water samples. We developed a species-specific qPCR assay targeting the cytochrome c oxidase subunit I (COI). The assay was tested in silico, in vitro (DNA extracted from tissue) and in vivo (DNA extracted from water samples). Water samples were collected from five ponds with known Natterjack toad presence or absence to validate the sensitivity and specificity of the assay. The assay was shown to be highly specific to the Natterjack toad and tested positive only against toad tissue samples and eDNA samples from ponds with known species presence. We believe this method can be used for rapid assessment of species occurrence.

Histopathologic lesions in conventional pigs experimentally infected with Haemophilus parasuis serovar 5

2015 ◽  
Vol 43 (02) ◽  
pp. 91-96 ◽  
Author(s):  
R.-L. Austin-Busse ◽  
A. Ladinig ◽  
G. Balka ◽  
S. Zoels ◽  
M. Ritzmann ◽  
...  
Keyword(s):  
Clinical Signs ◽  
Control Group ◽  
Post Inoculation ◽  
Haemophilus Parasuis ◽  
Tissue Samples ◽  
Group A ◽  
Pathologic Lesions ◽  
Group B ◽  
Kidney Lesions ◽  
Challenge Group

Summary Objective: In the present study various tissues of pigs were investigated for the presence of histopathologic lesions after an experimental infection with Haemophilus (H.) parasuis serovar 5. Material and methods: Conventional pigs (n = 36) were divided into a control group B (n = 9) and a challenge group A (n = 27), which was infected intratracheally. Pigs that did not die prior to study termination were euthanized on day 14 post inoculation. Postmortem samples of the lung, heart, liver, kidney, spleen, left tarsal joint capsule and brain were collected. Results: All but one pig with detectable histopathologic lesions (n = 11) showed typical macroscopic changes. Histopatho logic examination of all tissue samples identified pyelitis (n = 10), synovitis (n = 7) and meningitis (n = 7) and all those animals were euthanized prior to study termination. No histopathologic lesions were found in pigs of the control group. The correlations between pyelitis and meningitis, pyelitis and synovitis and synovitis and meningitis were significant (p < 0.001). No significant correlation could be observed between the histopathologic and the clinical examination of the joints. The investigation of samples from the joints by PCR was not significantly correlated with the observed synovitis. The clinical observation of neurologic signs was significantly correlated with meningitis (p = 0.03). A significant correlation (p < 0.001) could be detected between meningitis and the detection of H. parasuis by PCR in brain samples. Conclusions: H. parasuis constantly causes clinical signs and pathologic lesions as soon as it infects the brain while it can infect the joints without causing histopathologic lesions. Pigs with histopathologic lesions do not always show typical clinical signs. Only few studies described the finding of kidney lesions in pigs with Glässer’s disease and this is the first study to describe a pyelitis in pigs experimentally infected with H. parasuis. The observed pyelitis mainly occurred in acute cases.

scholarly journals Pathogenicity of an African swine fever virus strain isolated in Vietnam and alternative diagnostic specimens for early detection of viral infection

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Hu Suk Lee ◽  
Vuong Nghia Bui ◽  
Duy Tung Dao ◽  
Ngoc Anh Bui ◽  
Thanh Duy Le ◽  
...  
Keyword(s):  
Early Detection ◽  
Oral Fluid ◽  
African Swine Fever Virus ◽  
Fluid Collection ◽  
Clinical Signs ◽  
African Swine Fever ◽  
Post Inoculation ◽  
Tissue Samples ◽  
Viral Loads ◽  
Specimen Collection

Abstract Background African swine fever (ASF), caused by the ASF virus (ASFV), was first reported in Vietnam in 2019 and spread rapidly thereafter. Better insights into ASFV characteristics and early detection by surveillance could help control its spread. However, the pathogenicity and methods for early detection of ASFV isolates from Vietnam have not been established. Therefore, we investigated the pathogenicity of ASFV and explored alternative sampling methods for early detection. Results Ten pigs were intramuscularly inoculated with an ASFV strain from Vietnam (titer, 103.5 HAD50/mL), and their temperature, clinical signs, and virus excretion patterns were recorded. In addition, herd and environmental samples were collected daily. The pigs died 5–8 days-post-inoculation (dpi), and the incubation period was 3.7 ± 0.5 dpi. ASFV genome was first detected in the blood (2.2 ± 0.8) and then in rectal (3.1 ± 0.7), nasal (3.2 ± 0.4), and oral (3.6 ± 0.7 dpi) swab samples. ASFV was detected in oral fluid samples collected using a chewed rope from 3 dpi. The liver showed the highest viral loads, and ear tissue also exhibited high viral loads among 11 tissues obtained from dead pigs. Overall, ASFV from Vietnam was classified as peracute to acute form. The rope-based oral fluid collection method could be useful for early ASFV detection and allows successful ASF surveillance in large pig farms. Furthermore, ear tissue samples might be a simple alternative specimen for diagnosing ASF infection in dead pigs. Conclusions Our data provide valuable insights into the characteristics of a typical ASFV strain isolated in Vietnam and suggest an alternative, non-invasive specimen collection strategy for early detection.

Studies on Diagnosis of Bovine Brucellosis by Immunocyto-chemistry and Immunohistochemistry

2021 ◽  
Author(s):  
K.S. Lakshmikanth ◽  
N.S. Sharma ◽  
D. Pathak ◽  
Paviter Kaur
Keyword(s):  
Biochemical Analysis ◽  
Placental Tissue ◽  
Diagnostic Techniques ◽  
Clinical Samples ◽  
Bovine Brucellosis ◽  
Reproductive Disorders ◽  
Chain Reaction ◽  
Tissue Samples ◽  
Polymerase Chain ◽  
Positive Results

Background: Brucellosis is a major threat to livestock economy and an important zoonotic disease. A rapid and accurate diagnosis is a necessity to curb the spread and progress of the disease. The current study aimed to evaluate sensitivity of Immunocytochemistry and Immunohistochemistry methods for detection of Brucella spp.Methods: A total of 50 samples comprising of fetal stomach content, vaginal discharges and placenta were collected from cattle and buffaloes suffering from abortions and other reproductive disorders in and around Ludhiana, Punjab during the period 2017-2018. All the samples were processed for isolation and confirmed with biochemical analysis and Polymerase chain reaction (PCR). The isolates obtained and 43 clinical samples excluding placental samples were subjected to Immunocytochemistry (ICC). Immunohistochemistry (ICH) was performed on placental samples.Result: A total of four isolates were recovered from the screened samples. The four isolates also yielded positive results in Immunocytochemistry. Among the 43 clinical samples screened by Immunocytochemistry, five were positive, however only 3 isolates were recovered on isolation. A total of seven placental tissue samples were processed and subjected to immunohistochemistry. Of the three placental samples positive by immunohistochemistry, only one sample was isolated on culture. The results suggest that both immunocytochemistry and immunohistochemistry are sensitive diagnostic techniques in comparison to isolation.

Patch occupancy and activity pattern of the spotted paca (Cuniculus paca Linnaeus, 1766) in a protected area of the Atlantic Forest, Brazil

Mammalia ◽  
2019 ◽  
Vol 83 (4) ◽  
pp. 363-371 ◽  
Author(s):  
Atilla C. Ferreguetti ◽  
Walfrido M. Tomas ◽  
Helena G. Bergallo
Keyword(s):  
Atlantic Forest ◽  
Extreme Temperature ◽  
Temporal Distribution ◽  
Camera Traps ◽  
Activity Period ◽  
Spatial And Temporal Distribution ◽  
Patch Occupancy ◽  
Activity Frequency ◽  
Palm Species ◽  
Species Activity

Abstract The spotted paca Cuniculus paca (Linnaeus, 1766) is a medium-sized caviomorph rodent of the Cuniculidae family that mainly inhabits tropical forests, but may occur in other habitat types, often associated with water bodies. We aimed to verify which factors influence the spatial and temporal distribution of C. paca in the Vale Natural Reserve (VNR), Espírito Santo, Brazil. We used 39 camera traps to model occupancy and detectability and to estimate the species activity period. The spotted paca showed high occupancy at low distances from water resources and high densities of palm species. The species avoided areas with high poaching intensity, and activity frequency was reduced by extreme temperature and by a higher intensity of poaching. We conclude that in the VNR, the C. paca is a nocturnal species and that it is necessary to assess other elements that could potentially affect the spatial and temporal distribution of the spotted paca in the Atlantic Forest.

scholarly journals Clinical, pathological and epidemiological aspects of outbreaks of bluetongue disease in sheep in the central region of Rio Grande do Sul

2017 ◽  
Vol 37 (12) ◽  
pp. 1443-1452
Author(s):  
Ronaldo M. Bianchi ◽  
Welden Panziera ◽  
Tatiane C. Faccin ◽  
Gisane L. de Almeida ◽  
Juliana F. Cargnelutti ◽  
...  
Keyword(s):  
Central Region ◽  
Striated Muscle ◽  
Clinical Signs ◽  
Rio Grande ◽  
Nasal Discharge ◽  
Rio Grande Do Sul ◽  
Sample Collection ◽  
Area At Risk ◽  
Tissue Samples ◽  
Facial Swelling

ABSTRACT: This article describes the clinical, pathological and epidemiological aspects of 17 outbreaks of bluetongue (BT) disease in sheep occurring between December 2014 and July 2015 in the central region of Rio Grande do Sul state (RS), southern Brazil. Affected farms were visited for clinical examination, necropsy, sample collection and epidemiological investigation. The outbreaks were seasonal and occurred during the summer and autumn. A total of 180 sheep (20.4%) out of 884 in 17 small herds were affected. All ages of Texel and mixed breed sheep were affected. However, lambs (younger than one year) had higher morbidity than adult sheep. The most frequent clinical signs were anorexia, lethargy, loss of body condition, facial swelling mainly involving the lips, and greenish seromucous or mucous nasal discharge. Pulmonary lesions characterized by edema were the most prevalent findings; however, erosive and ulcerative lesions in the upper gastrointestinal tract, as well as cardiac, skeletal muscle and esophageal striated muscle necrosis, and hemorrhage in the pulmonary artery were also frequent. The bluetongue virus (BTV) genome was detected by RT-PCR in blood and tissue samples (spleen and lungs) of 21 animals from 17 outbreaks. The virus involved in the outbreak 3 was subsequently isolated and shown to belong to serotype 17, for the first time reported in Brazil. In summary, our data support the BTV genotype 17 as the etiological agent of the outbreaks and indicate that the central region of RS is an area at risk for BT in sheep, a disease previously not recognized in the region.

scholarly journals A Simple Technique for the Identification of Environmental DNA (eDNA ) in the Water Samples

2021 ◽  
pp. 77-80
Author(s):  
Chitra K. Y.
Keyword(s):  
Water Samples ◽  
Large Scale ◽  
Dna Isolation ◽  
Simple Technique ◽  
Environmental Dna ◽  
Water Bodies ◽  
Easy Method ◽  
Selective Staining ◽  
Generation Sequencing ◽  
Dark Pink

The environmental DNA(eDNA) is the DNA that is shed by the organisms in their environment by different ways viz. , mucous, faeces, skin, eggs, sperms and also when these organisms die due to natural death or disease. The eDNA will persist for several days. Identification of eDNA is a useful method of determining the organisms present in an aquatic environment like amphibians, reptiles, fishes , insects and larval forms of some of these organisms. By analysing the e-DNA it is possible to monitor the species distribution in water bodies like lakes and ponds simply by collecting a sample of water. The technique can be applied for the survey of the water bodies on a large scale for the genomic, taxonomic as well as pollutional studies. The DNA isolation procedures that are available are laborious and time consuming. Therefore, during the present study, a simplified method was devised i. e. , isolation of eDNA with ethanol after which Feulgen stain was applied to identify and confirm it, as it is an easy method before proceeding to work with the isolated eDNA using other techniqnies for further studies. The Feulgen method is used for the selective staining and the localisation of the DNA in the tissues but is adopted during the present study for the water samples for quick identification of eDNA. The smear of eDNA stained with Feulgen showed dark pink or magenta colour under the microscope where it was concentrated but stained lightly when dispersed and fragmented as observed in the present study. Further studies of the isolated eDNA are in progress in our laboratory for quantifying and sequencing eDNA using latest techniques like next generation sequencing for the identification of fish species in the lakes.

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