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Major depressive disorder (MDD) is a serious disease and a burden to patients, families and society. Rodent experiments and human studies suggest that several neuropeptide systems are involved in mood regulation. The aim of this study is two-fold: (i) to monitor, with qPCR, transcript levels of the substance P/tachykinin (TAC), NPY and CCK systems in bulk samples from control and suicide subjects, targeting five postmortem brain regions including locus coeruleus (LC); and (ii) to analyse expression of neuropeptide family transcripts in LC neurons of 'normal' postmortem brains by using laser capture microdissection with Smart-Seq2 RNA sequencing. qPCR revealed distinct regional expression patterns in male and female controls with higher levels for the TAC system in the dorsal raphe nucleus and LC, versus higher transcripts levels of the NPY and CCK systems in prefrontal cortex. In suicide patients, TAC, TAC receptors and a few NPY family transcript levels were increased mainly in prefrontal cortex and LC. The second study on 'normal' noradrenergic LC neurons revealed expression of transcripts for GAL, NPY, TAC1, CCK, and TACR1 and many other peptides (e.g. Cerebellin4 and CARTPT) and receptors (e.g. Adcyap1R1 and GPR173). These data and our previous results on suicide brains indicates that the tachykinin and galanin systems may be valid targets for developing antidepressant medicines. Moreover, the perturbation of neuropeptide systems in MDD patients, and the detection of further neuropeptide and receptor transcripts in LC, shed new light on signalling in noradrenergic LC neurons and on mechanisms possibly associated with mood disorders., QC 20231218

Abstrakt Bakgrund: Barn som drabbas med tumörer behöver genomgå strålbehandling. För att uppnå optimala behandlingsresultat krävs sederingar eller generella anestesi. Tidigare studier har indikerat att strålningsspecifika effekter påverkade kognitiva funktioner hos barn. Syfte: Karakterisera biomarkörer för skador orsakade av strålning och undersöka de neurotoxiska effekterna som uppstår vid hög exponering för gammastrålning. Metod: Del1: Design av 8 primrar för musgenerna Hes1, Hes5, Timp1, Aldh11l1 . RNA-pool används från fem olika grupper. QPCR-metoden används för att bestämma effektiviteten hos de 8 primrarna. Del2: Experimenten utfördes på NMRI-möss på postnatal dag 10. Genuttryck undersöktes för 5 kontrollgrupper och 5 exponerade grupper för hög dos gammastrålning 2Gy. DNA-extraktion utfördes på hippocampus och prefrontalcortex från mössens hjärna. qPCR-metoden används för att studera uppreglering och nedreglering av gener. Resultat: Effektiviteten för primrarna Hes 5 = 80%, Hes1:2= 93% vilket uppfyllde kriterierna 80–110%. Resultat för genererad nedreglering av BDNF i hippocampus och uppreglering av Hes 5 och Jagged-1 i prefrontal cortex, vilket visade en signifikant skillnad mellan gruppernas medelvärden. Däremot observerade ingen signifikant skillnad i uttrycket av generna Nrf2, Grin2, Jagged-1 i hippocampus. Slutsats: Strålningsdoser som används för att behandla hjärntumörer hos barn kan påverka hur vissa allmänna uttryck och leda till dynamiska förändringar i uttrycket som styr normal hjärnutveckling.

In populations of healthy show horses, the subclinical transmission and circulation of respiratory pathogens can lead to disease outbreaks. Due to recent outbreaks of equine herpesvirus-1 myeloencephalopathy (EHM) in the USA and Europe, many show organizers have instituted various biosecurity protocols such as individual horse testing, monitoring for early clinical disease and increasing hygiene and cleanliness protocols. The aim of this study was to determine the accuracy of detecting EHV-1 in the various environmental samples collected from the stalls of subclinical shedders. Four healthy adult horses were vaccinated intranasally with a modified-live EHV-1 vaccine in order to mimic subclinical shedding. Three additional horses served as non-vaccinated controls. All the horses were stabled in the same barn in individual stalls. Each vaccinated horse had nose-to-nose contact with at least one other horse. Prior to the vaccine administration, and daily thereafter for 10 days, various samples were collected, including a 6 rayon-tipped nasal swab, an environmental sponge, a cloth strip placed above the automatic waterer and an air sample. The various samples were processed for nucleic acid purification and analyzed for the presence of EHV-1 via quantitative PCR (qPCR). EHV-1 in nasal secretions was only detected in the vaccinated horses for 1-2 days post-vaccine administration. The environmental sponges tested EHV-1 qPCR-positive for 2-5 days (median 3.5 days) in the vaccinated horses and 1 day for a single control horse. EHV-1 was detected by qPCR in stall strips from three out of four vaccinated horses and from two out of three controls for only one day. EHV-1 qPCR-positive air samples were only detected in three out of four vaccinated horses for one single day. For the vaccinated horses, a total of 25% of the nasal swabs, 35% of the environmental stall sponges, 7.5% of the strips and 7.5% of the air samples tested qPCR positive for EHV-1 during the 10 study days. Wh

Most pneumococcal disease occurs among infants and older adults and is thought to be driven by the transmission of Streptococcus pneumoniae from young children to these vulnerable age groups. However, pneumococcal disease outbreaks also affect non-elderly adults living or working in congregate, close-contact settings. Little is known about pneumococcal carriage in such populations. From July to November 2020, we collected saliva from low-income adult farmworkers in Monterey County, California, and tested for pneumococcal carriage following culture enrichment via quantitative PCR assays targeting the pneumococcal lytA and piaB genes. Participants were considered to carry pneumococci if lytA and piaB cycle threshold values were both below 40. Among 1,283 participants enrolled in our study, 117 (9.1%) carried pneumococci. Carriers tended more often than non-carriers to be exposed to children aged <5 years [odds ratio (OR) = 1.45 (0.95-2.20)] and overcrowding [OR = 1.48 (0.96-2.30) and 2.84 (1.20-6.73), respectively, for participants in households with >2-4 and >4 persons per bedroom vs ≤2 persons per bedroom]. Household overcrowding remained associated with increased risk of carriage among participants not exposed to children aged <5 years [OR = 2.05 (1.18-3.59) for participants living in households with >2 vs ≤2 persons per bedroom]. Exposure to children aged <5 years and overcrowding were each associated with increased pneumococcal density among carriers [piaB cT difference of 2.04 (0.36-3.73) and 2.44 (0.80-4.11), respectively]. While exposure to young children was a predictor of pneumococcal carriage, associations of overcrowding with increased prevalence and density of carriage in households without young children suggest that transmission also occurs among adults in close-contact settings.IMPORTANCEAlthough infants and older adults are the groups most commonly affected by pneumococcal disease, outbreaks are known to occur among healthy, working

Den nationella plastkartläggningen visade att år 2017 användes över 813 miljoner engångsprodukter av plast inom hälso- och sjukvården i Sverige. Den största delen av detta avfall hanteras genom förbränning, och för att minska den miljöpåverkan detta leder till har ett antal initiativ tagits för att inkludera materialet i slutna kretslopp, där plasten används i samma applikation på nytt och där materialets höga kvalité kan behållas. För att kunna göra det på ett säkert sätt krävs dock att materialet kan steriliseras för att vara säkert att hantera. Detta projekt har haft som mål att ta fram en metod för att mäta den eventuella steriliseringsprocess som sker när plast återvinns i den så kallade regranuleringsprocessen. Denna process innebär att plasten mals ner och smälts vid omkring 200°C. Om denna process är effektiv för att sterilisera materialet behövs inget separat steriliseringssteg innan regranuleringen. Vi har i projektet tillsatt bakterien Bacillus atrophaeus, en organism som ofta används för verifiering av sterilisering, till regranuleringen av LDPE-plast. Vi har sedan utvärderat olika metoder för att extrahera DNA från det regranulerade materialet samt utvecklat en qPCR-metod för att kvantifiera antalet överlevande bakterier. På så sätt har vi försökt mäta den eventuella steriliseringen av det regranulerade plastmaterialet.Resultatet visar att det fortfarande finns svårigheter att frigöra DNA-molekylerna från plasten, och att detta steg även kan ha en negativ påverkan på DNA-kvalitén. qPCR-metoden kan detektera B. atrophaeus och visar i ett separat värmeexperiment på en förväntad steriliseringseffekt över tid. Denna effekt kunde dock inte reproduceras i de labbförsök som utfördes med extruder (en utrustning som används för att pressa smält plastgranulat till den profil som önskas). Ett antal möjliga förklaringar till detta, samt förslag till vidare forskning presenteras avslutningsvis i rapporten. Exempel på fortsatta forskningsfrågor som presenteras är

Seasonal floodplains in the Amazon basin are important sources of methane (CH4), while upland forests are known for their sink capacity. Climate change effects, including shifts in rainfall patterns and rising temperatures, may alter the functionality of soil microbial communities, leading to uncertain changes in CH4 cycling dynamics. To investigate the microbial feedback under climate change scenarios, we performed a microcosm experiment using soils from two floodplains (i.e., Amazonas and Tapajós rivers) and one upland forest. We employed a two-factorial experimental design comprising flooding (with non-flooded control) and temperature (at 27 °C and 30 °C, representing a 3 °C increase) as variables. We assessed prokaryotic community dynamics over 30 days using 16S rRNA gene sequencing and qPCR. These data were integrated with chemical properties, CH4 fluxes, and isotopic values and signatures. In the floodplains, temperature changes did not significantly affect the overall microbial composition and CH4 fluxes. CH4 emissions and uptake in response to flooding and non-flooding conditions, respectively, were observed in the floodplain soils. By contrast, in the upland forest, the higher temperature caused a sink-to-source shift under flooding conditions and reduced CH4 sink capability under dry conditions. The upland soil microbial communities also changed in response to increased temperature, with a higher percentage of specialist microbes observed. Floodplains showed higher total and relative abundances of methanogenic and methanotrophic microbes compared to forest soils. Isotopic data from some flooded samples from the Amazonas river floodplain indicated CH4 oxidation metabolism. This floodplain also showed a high relative abundance of aerobic and anaerobic CH4 oxidizing Bacteria and Archaea. Taken together, our data indicate that CH4 cycle dynamics and microbial communities in Amazonian floodplain and upland forest soils may respond differentl

Vitt snus ökande popularitet har skapat oro för dess påverkan på immunförsvaret. I föreliggande studie undersöks nikotinets påverkan på uttrycket av interleukin-2-receptorn alfa (IL-2R-alfa eller CD25) hos CD4+ T-celler under in vitro-stimulering. Tidigare studier indikerar negativa effekter såsom minskat antingen upptag hos antigenpresenterande celler, cytokinproduktion och uttryck av receptorer som CD28 och CTLA-4, vilka är viktiga för CD4+ T-cellernas aktivering, differentiering samt bromsning av aktiveringen. Andra studier har visat att alfa-4- samt alfa-7-nikotinerg acetylkolinreceptorer uttrycks på CD4+ T-celler, vilket i sin tur har visat sig påverka cytokin produktionen i närvaro av nikotin under stimulering. CD25 är en viktig receptor vid aktiveringen av T-celler och är överuttryckt vid exempelvis akut T-cells leukemi (ATL). Målet med denna studie är att förstå hur nikotin kan påverka CD25-uttrycket och därmed påverka immunförsvaret och effektiviteten av terapier. Med hjälp av flödescytometri och kvantitativ polymeraskedjereaktion (qPCR) kunde en tydlig skillnad detekteras mellan nikotin/PHA-stimulering och enbart PHA-stimulering hos en donator jämfört med de andra donatorerna. Metoderna inkluderar cellseparation, stimulering med fytohemagglutinin (PHA) i närvaro av nikotin, analys av uttrycket CD25 med flödescytometri och analys av genuttryck av CD25 med qPCR. Resultaten visade varierande svar mellan tre donatorer, med en tydlig skillnad mellan nikotin/PHA- och enbart PHA-stimulering för en donator jämfört med de andra donatorerna. Denna studie pekar på potentiella effekter av nikotin på immunförsvaret och betonar behovet av vidare undersökningar för att förstå mekanismerna bakom detta., The increasing popularity of white snus has raised concerns about its impact on the immune system. The present study investigates the effect of nicotine on the expression of interleukin-2 receptor alpha (IL-2R-alpha or CD25) in CD4+ T cells during in vitro stimulation. Previous studies have indicated negative effects such as decreased uptake by antigen-presenting cells, cytokine production, and expression of receptors such as CD28 and CTLA-4, which are important for the activation, differentiation, and inhibition of CD4+ T cell activation. Other studies have shown that alpha-4 and alpha-7 nicotinic acetylcholine receptors are expressed on CD4+ T cells, which in turn has been shown to affect cytokine production in the presence of nicotine during stimulation. CD25 is an important receptor for T cell activation and is overexpressed in conditions such as acute T cell leukemia (ATL). The aim of this study is to understand how nicotine may affect CD25 expression and thereby influence the immune response and the effectiveness of therapies. Using flow cytometry and quantitative polymerase chain reaction (qPCR), a clear difference was detected between nicotine/PHA stimulation and PHA stimulation alone in one donor compared to the other donors. The methods include cell separation, stimulation with phytohemagglutinin (PHA) in the presence of nicotine, analysis of CD25 expression by flow cytometry and analysis of CD25 gene expression by qPCR. The results showed varying responses among three donors, with a clear difference between nicotine/PHA and PHA stimulation alone for one donor compared to the others. This study highlights potential effects of nicotine on the immune system and underscores the need for further investigations to understand the mechanisms behind this.

Population numbers of bumblebees has been on a steady decline leading to the endangerment of several species that are critical to the growth and pollination of crops and plants. Varying factors have contributed to this situation, one key factor being pathogens wiping out colonies. Studying pathogens that diminish bumblebee populations is vital to understanding how to combat the decline of these important species effectively. The aim of this study was to analyse five of the most common pathogens that exist in the abdomen of five different species of bumblebees. This was achieved by extracting DNA, from 176 previously acquired frozen samples, through homogenisation and varying washing steps before an analysis of the resulting nucleic acids. The analysis was done with quantitative polymerase chain reactions to quantify the amounts of each pathogen, as well as a passive reference nucleic acid, to then be able to calculate the absolute total -amount of pathogen carried by each individual bumblebee. The results showed that almost half of the bumblebees were infected by Nosema, one sixth were infected by Crithidia and Apicystis while only a small number of individuals had been infected by Bombus Densovirus and the Apis Mellifera Filamentous Virus. Bombus Lapponicus was the most infected species. The conclusion based on the results is that future research could put a focus on the more commonly found parasites Nosema, Crithidia and Apicystis. Additional research should also be undertaken into the factors contributing to the less common pathogens Bombus Densovirus and the Apis Mellifera Filamentous Virus.

Effective conservation of endangered species depends on knowledge of their distributions, but species detection can often be challenging. An example of this is provided by the Critically Endangered Margaret River burrowing crayfish (Engaewa pseudoreducta), which is highly cryptic. Due to the burrowing habit of this crayfish, detection of this species currently requires a great deal of effort, the results are often non-conclusive, and, as it involves manual excavation of their burrows, the habitat of this and other species is destroyed in the detection process. In response to these challenges, this study developed and optimized a species-specific probe-based qPCR assay targeting the 16S gene region to detect the target species from environmental samples. Three test substrates—chimney pellets (soil expelled by a crayfish as it digs its burrow), burrow scrapes (soil lining the inside of a burrow), and burrow water (water that is filling the burrow space)—were tested from 11 crayfish burrows thought to have been constructed by the target species to see if eDNA could be detected. DNA from the target species was successfully amplified from both chimney pellets (6/11 samples) and burrow scrapes (3/11 samples); however, E. pseudoreducta was not detected in any water samples. As previously stated, sampling techniques to confirm the presence of this species have relied on burrow excavation (resulting in habitat destruction) and were often not definitive; therefore, replacing the traditional survey method with a noninvasive eDNA-based technique could be of enormous benefit to the management and conservation of this (and similar) species.

The monk seal is the most endangered pinniped in the world and the only one found in the Mediterranean, where its distribution and abundance have suffered a drastic decline in the last few decades. Data on its status are scattered due to both its rarity and evasiveness and records are biased towards occasional, mostly coastal encounters. Nowadays, molecular techniques allow us to detect and quantify minute amounts of DNA traces released into the environment (eDNA) by any organism. A species-specific molecular assay is now available for detecting the recent presence of the monk seal in the water column through the analysis of sea-water samples collected from the sea surface. The project “Spot the Monk” uses this non-invasive detection tool to monitor monk seal occurrence in Mediterranean waters by means of eDNA analysis. The simplicity in the acquisition of samples together with the need to collect samples in multiple points simultaneously made the project well suited to the involvement of the general public. Up to today, about 350 samples have been collected and analysed in the central-western Mediterranean by researchers and a multifarious range of citizen scientists - from recreational sailing organisations, both amateur and competitive sportsmen, to fishermen. This work announces the launch of an open-source Observatory (https://www.spot-themonk- observatory.com/) where the project outcomes are publicly accessible as soon as they are produced. Embracing the principles of Open Science, we believe that such an approach can contribute to filling the knowledge gap about the distribution of this charismatic species in our seas, providing, at the same time, a proof of concept on how data collected by a variety of actors can be returned to the scientific and non-scientific communities in an innovative format for immediate consultation.

Legionella pneumophila est la cause d’épidémies où les victimes présentent de la fièvre de Pontiac et la légionellose avec une mortalité significative. Les origines de ces épidémies se retrouvent dans les systèmes d’eau (majoritairement les tours de refroidissement). Pour pallier à ce problème, le besoin s’est intensifié d’un biosenseur précis et rapide des eaux industrielles et environnementales. Bien que d’autres méthodes existent pour la détection, elles ont les défauts de prendre beaucoup de temps, d’être techniquement complexes et d’exiger de la main-d’œuvre expérimentée. Le biosenseur développé viendra résoudre ses désavantages. Dans ce mémoire est présenté l’article publié évaluant des anticorps polyclonaux anti-L. pneumophila et des anticorps recombinants anti-lipopolysaccharide de L. pneumophila. Grâce à la technique d’immunoessai combiné à une quantification par PCR, nous avons démontré que l’anticorps recombinant capture un tiers de moins de L. pneumophila vivantes que l’anticorps polyclonal, mais a une réduction de 40% dans son écart-type entre les essais. Ces résultats étaient maintenus dans les expériences avec des solutions de bactéries individuelles et des solutions de bactéries cibles mélangées avec des bactéries non-cibles. La haute réplicabilité des anticorps recombinants (même comparé à des anticorps polyclonaux provenant d’un seul lot) indique le meilleur choix de caractéristiques pour un appareil comme un biosenseur. La comparaison semblable des caractéristiques de sensibilité et de spécificité des anticorps recombinants versus les anticorps polyclonaux octroie de grandes opportunités aux anticorps recombinants pour leur développement dans les futures avancées technologiques et biologiques.

Pyocyanin is a blue-green phenazine pigment and one of the most virulent factors produced by the opportunistic pathogen Pseudomonas aeruginosa. It has a redox activity and a toxic impact on living cells, as it interacts with oxygen to produce reactive oxygen species (ROS). Using antibiotics at a sub-lethal dose has an unexpected influence on the expression of pyocyanin-producing genes. In this study, qPCR technique was performed to identify the effect of eight antibiotics (cefotaxime, ampicillin, amoxiclav, ceftazidime, ceftriaxone, chloramphenicol, kanamycin and tetracycline) on the gene expression level of pyocyanin synthetic genes in P. aeruginosa isolated from different clinical sources of a few hospitals in Mosul, Iraq using qPCR technique. It was found that when P. aeruginosa was grown in media containing cefotaxime (CTX 30 µg/mL), ampicillin (AM 25 µg/mL) or amoxiclav (AMC 30 µg/mL), up-regulated the expression of pyocyanin producing genes belonging to different operons thereby increased pyocyanin production. Overexpression occurred in (CTX) treatment in PhzA1 operon with 235.56 fold change and phzM and phzS genes with 340.14, 280.13 fold change, respectively. Lower expression levels showed in tetracycline (TE 30 µg/mL) treatment, which was a (1.44) fold change for phzA1 and a (1.64, 1.08) fold change for phzM and phzS genes. More caution should be considered when delivering antibiotics to treat P. aeruginosa infections, as using drugs that the bacteria resists or at sub-lethal concentrations may trigger up-regulation of virulence factors, aiding in the spread of the disease.

BACKGROUND: Malaria remains a significant cause of morbidity and mortality in Ethiopia with an estimated 3.8 million cases in 2021 and 61% of the population living in areas at risk of malaria transmission. Throughout the country Plasmodium vivax and Plasmodium falciparum are co-endemic, and Duffy expression is highly heterogeneous. The public health significance of Duffy negativity in relation to P. vivax malaria in Ethiopia, however, remains unclear. This study seeks to explore the prevalence and rates of P. vivax malaria infection across Duffy phenotypes in clinical and community settings. METHODS: A total of 9580 and 4667 subjects from community and health facilities from a malaria endemic site and an epidemic-prone site in western Ethiopia were enrolled and examined for P. vivax infection and Duffy expression from February 2018 to April 2021. Association between Duffy expression, P. vivax and P. falciparum infections were examined for samples collected from asymptomatic community volunteers and symptomatic subjects from health centres. RESULTS: Infection rate of P. vivax among Duffy positives was 2-22 fold higher than Duffy negatives in asymptomatic volunteers from the community. Parasite positivity rate was 10-50 fold higher in Duffy positives than Duffy negatives among samples collected from febrile patients attending health centres and mixed P. vivax and P. falciparum infections were significantly more common than P. vivax mono infections among Duffy negative individuals. Plasmodium vivax parasitaemia measured by 18sRNA parasite gene copy number was similar between Duffy positives and Duffy negatives. CONCLUSIONS: Duffy negativity does not offer complete protection against infection by P. vivax, and cases of P. vivax in Duffy negatives are widespread in Ethiopia, being found in asymptomatic volunteers from communities and in febrile patients from health centres. These findings offer evidence for consideration when developing control and intervention strategie

Objectives: The impact of implant surfaces in dental biofilm development is presently unknown. The aim of this investigation was to assess in vitro the development of a complex biofilm model on titanium and zirconium implant surfaces, and to compare it with the same biofilm formed on hydroxyapatite surface. Methods: Six standard reference strains were used to develop an in vitro biofilm over sterile titanium, zirconium and hydroxyapatite discs, coated with saliva within the wells of pre-sterilized polystyrene tissue culture plates. The selected species used represent initial (Streptococcus oralis and Actinomyces naeslundii), early (Veillonella parvula), secondary (Fusobacterium nucleatum) and late colonizers (Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans). The developed biofilms (growth time 1 to 120h) were studied with confocal laser scanning microscopy using a vital fluorescence technique and with low-temperature scanning electron microscopy. The number (colony forming units/biofilm) and kinetics of the bacteria within the biofilm were studied with quantitative PCR (qPCR). As outcome variables, the biofilm thickness, the percentage of cell vitality and the number of bacteria were compared using the analysis of variance. Results: The bacteria adhered and matured within the biofilm over the three surfaces with similar dynamics. Different surfaces, however, demonstrated differences both in the thickness, deposition of the extracellular polysaccharide matrix as well as in the organization of the bacterial cells. Significance: While the formation and dynamics of an in vitro biofilm model was similar irrespective of the surface of inoculation (hydroxyapatite, titanium or zirconium), there were significant differences in regards to the biofilm thickness and three-dimensional structure., Depto. de Especialidades Clínicas Odontológicas, Fac. de Odontología, TRUE, pub

In the global context of seawater deoxygenation triggered by climate change and anthropogenic activities, changes in redox gradients impacting biogeochemical transformations of pollutants, such as mercury, become more likely. Being the largest anoxic basin worldwide, with high concentrations of the potent neurotoxic methylmercury (MeHg), the Black Sea is an ideal natural laboratory to provide new insights about the link between dissolved oxygen concentration and hgcAB gene-carrying (hgc+) microorganisms involved in the formation of MeHg. We combined geochemical and microbial approaches to assess the effect of vertical redox gradients on abundance, diversity, and metabolic potential of hgc+ microorganisms in the Black Sea water column. The abundance of hgcA genes [congruently estimated by quantitative PCR (qPCR) and metagenomics] correlated with MeHg concentration, both maximal in the upper part of the anoxic water. Besides the predominant Desulfobacterales, hgc+ microorganisms belonged to a unique assemblage of diverse—previously underappreciated—anaerobic fermenters from Anaerolineales, Phycisphaerae (characteristic of the anoxic and sulfidic zone), Kiritimatiellales, and Bacteroidales (characteristic of the suboxic zone). The metabolic versatility of Desulfobacterota differed from strict sulfate reduction in the anoxic water to reduction of various electron acceptors in the suboxic water. Linking microbial activity and contaminant concentration in environmental studies is rare due to the complexity of biological pathways. In this study, we disentangle the role of oxygen in shaping the distribution of Hg-methylating microorganisms consistently with MeHg concentration, and we highlight their taxonomic and metabolic niche partitioning across redox gradients, improving the prediction of the response of marine communities to the expansion of oxygen-deficient zones. IMPORTANCE Methylmercury (MeHg) is a neurotoxin detected at high concentrations in certain marine ecosyst

In the global context of seawater deoxygenation triggered by climate change and anthropogenic activities, changes in redox gradients impacting biogeochemical transformations of pollutants, such as mercury, become more likely. Being the largest anoxic basin worldwide, with high concentrations of the potent neurotoxic methylmercury (MeHg), the Black Sea is an ideal natural laboratory to provide new insights about the link between dissolved oxygen concentration and hgcAB gene-carrying (hgc+) microorganisms involved in the formation of MeHg. We combined geochemical and microbial approaches to assess the effect of vertical redox gradients on abundance, diversity, and metabolic potential of hgc+ microorganisms in the Black Sea water column. The abundance of hgcA genes [congruently estimated by quantitative PCR (qPCR) and metagenomics] correlated with MeHg concentration, both maximal in the upper part of the anoxic water. Besides the predominant Desulfobacterales, hgc+ microorganisms belonged to a unique assemblage of diverse—previously underappreciated—anaerobic fermenters from Anaerolineales, Phycisphaerae (characteristic of the anoxic and sulfidic zone), Kiritimatiellales, and Bacteroidales (characteristic of the suboxic zone). The metabolic versatility of Desulfobacterota differed from strict sulfate reduction in the anoxic water to reduction of various electron acceptors in the suboxic water. Linking microbial activity and contaminant concentration in environmental studies is rare due to the complexity of biological pathways. In this study, we disentangle the role of oxygen in shaping the distribution of Hg-methylating microorganisms consistently with MeHg concentration, and we highlight their taxonomic and metabolic niche partitioning across redox gradients, improving the prediction of the response of marine communities to the expansion of oxygen-deficient zones. IMPORTANCE Methylmercury (MeHg) is a neurotoxin detected at high conc

Aquatic organisms are constantly at risk of being exposed to potentially harmful chemical compounds of natural or anthropogenic origin. Biological life can for instance respond to chemical stressors by changes in gene expression, and thus, certain gene transcripts can potentially function as biomarkers, i.e. early warnings, of toxicity and chemical stress. A major challenge for biomarker application is the extrapolation of transcriptional data to potential effects at the organism level or above. Importantly, successful biomarker use also requires basal understanding of how to distinguish actual responses from background noise. The aim of this thesis is, based on response magnitude and variation, to evaluate the biomarker potential in a set of putative transcriptional biomarkers of general toxicity and chemical stress. Specifically, I addressed a selection of six transcripts involved in cytoprotection and oxidative stress: catalase (cat), glutathione-S-transferase (gst), heat shock proteins 70 and 90 (hsp70, hsp90), metallothionein (mt) and superoxide dismutase (sod). Moreover, I used metal exposures to serve as a proxy for general chemical stress, and due to their ecological relevance and nature as sedentary filter-feeders, I used bivalves as study organisms. In a series of experiments, I tested transcriptional responses in the freshwater duck mussel, Anodonta anatina, exposed to copper or an industrial wastewater effluent, to address response robustness and sensitivity, and potential controlled (e.g. exposure concentration) and random (e.g. gravidness) sources of variation. In addition, I performed a systematic review and meta-analysis on transcriptional responses in metal exposed bivalves to (1) evaluate what responses to expect from arbitrary metal exposures, (2) assess the influence from metal concentration (expressed as toxic unit), exposure time and analyzed tissue, and (3) address potential impacts from publication bias in the scientific literature. Response

Masterarbeit Universität Innsbruck 2023

Bakgrund: För att barn inte ska behöva känna oro vid tumörterapi blir de nedsövda med anestetikum. Vid behandling av tumörer används joniserande strålning. Dock har tidigare studier funnit att exponering av joniserande strålning hos barn kan vara en bidragande faktor till kognitiva dysfunktioner. Man har också sett en synergistisk effekt mellan anestetikum och joniserande strålning. Syfte: Syftet med denna studie är att undersöka samverkanseffekten mellan olika anestetika och joniserande strålning. Metod: NMRI möss exponerades på postnatal dag 10. Totalt var det 6 exponeringsgrupper. Mössen exponerades antingen med ketamin (7,5 mg/kg) eller propofol (20 mg/kg). Individerna som exponerades för strålning (200 mGy) fick detta 1 timme efter administration av singeldos anestetikum. Provtagningen skedde 24 timmar efter exponering. Metoden qPCR användes för att undersöka uppregleringen och nedregleringen av gener. Resultat: En korrelation kunde observeras för generna Keap1 & Nrf2, BDNF & Grin2b och Jagged-1 & Hes5 genom att studera Spearmans korrelationskonstant. Inga signifikanta resultat erhölls för generna som studerades (p <0,05). Medelvärdena visade ingen signifikant förändring i relativ FC. Slutsats: Kombinationsbehandling med anestetikum + joniserande strålning visade inga större skillnader mellan exponeringsgrupperna i motsats till tidigare genomförda studier. Detta kan bero på tiden mellan exponering och provtagning. Det finns en korrelation mellan gener som har en teoretisk sammankoppling.

Background: Rickettsia helvetica, a spotted fever rickettsia, is transmitted to humans via ticks in Europe, North Africa, and Asia. The central nervous system is a crucial target for rickettsial diseases, which has been reported for 12 of the 31 species, of which R. helvetica is one. This study aimed, in an experimental model, to identify characteristics of R. helvetica infection in a mouse neuronal cell line, NSC-34. Results: NSC-34, a fusion cell line of mouse motor spinal cord neurons and neuroblastoma cells, was used as a model. Propagation of R. helvetica in neurons was confirmed. Short actin tails were shown at the polar end of the bacteria, which makes it likely that they can move intracellularly, and even spread between cells. Another protein, Sca4, which with the cell adhesion protein vinculin enables the passage of the cell membrane, was expressed during infection. No significant increase in TNF alpha levels was seen in the infected neurons, which is of interest because TNF alpha protects the host cell from infection-induced apoptotic death which is crucial for host cell survival. The bacteria were also shown to invade and grow in the cell nucleus of the neuron. Conclusions: The findings suggest that a R. helvetica infection may be harmful to NSC-34 neurons under these in vitro conditions, but the full effects of the infection on the cell need to be studied further, also on human neurons, to also understand the possible significance of this infection in relation to pathogenetic mechanisms.

På huden lever en sammansättning av olika mikroorganismer såsom bakterier, svampar och virus. Dessa mikroorganismer kallas hudens mikrobiom. Sammansättningen av en individs mikrobiom kan ge mycket information om en individens hälsa. För att undersöka sammansättningen av bakterier på hudytan med exempelvis qPCR, behöver bakterier samlas in och DNA extraheras. Bakteriekoncentrationen på hudens torrare områden som exempelvis armar har normalt en relativt låg bakteriekoncentration vid 102-104 bakterier per cm2. Huden koloniseras till stor del av grampositiva bakterier. Grampositiva bakterier är i regel svårare att lysera än andra bakterier och kräver därför hårdare lysering. En bra extraktionsmetod ska erhålla mycket DNA utan att påverka dess kvalité. I detta arbete utvärderades initialt fem olika extraktionsmetoder på bakteriesuspension med Staphylococcus aureus (S. aureus), både direkt på bakteriesuspension men också från svabb. Utvärdering gjordes på PureLink Microbiome DNA Purification Kit, QlAamp PowerFecal Pro, QlAamp DNA Mini Kit och KOH-EDTA. Metoden med QlAamp DNA Mini Kit testades med två olika protokoll och räknades som två separata metoder. Metoderna som gav bäst resultat vid initial utvärdering var PureLink Microbiome och KOH-EDTA. Därefter utvärderades dessa två metoderna på prov insamlat med svabb från huden på 10 frivilliga deltagare.

Introduction: Lactobacilli may prevent broilers from colonization with Campylobacter spp. and other gram-negative zoonotic bacteria through lactic acid production and modulation of the intestinal microbiota. This study evaluated the effects of daily intake of Lactiplantibacillus plantarum 256 (LP256) on Campylobacter jejuni (C. jejuni) loads in ceca and feces of C. jejuni challenged broilers, together with the changes in the gut microbiota. Methods: Two experiments were conducted using the broilers Ross 308 (R-308; Experiment 1) for 42 days and Rowan Ranger broilers (RR; Experiment 2) for 63 days. The LP256 strain was administered either via silage inoculated with LP256 or direct supplementation in the drinking water. Concurrently, haylage as a forage similar to silage but without any inoculum was tested. C. jejuni loads in fecal matter and cecal content were determined by plate counts and qPCR, respectively. The cecal microbiota, in response to treatments and the challenge, were assessed by 16S rRNA sequencing. Results and Discussion: Culturing results displayed a significant reduction in C. jejuni colonization (2.01 log) in the silage treatment in comparison to the control at 1 dpi (day post-infection) in Experiment 1. However, no treatment effect on C. jejuni was observed at the end of the experiment. In Experiment 2, no treatment effects on C. jejuni colonization were found to be statistically significant. Colonization load comparison at the peak of infection (3 dpi) to that at the end of the trial (32 dpi) revealed a significant reduction in C. jejuni in all groups, regardless of treatment. Colonization dynamics of C. jejuni in the cecal samples analyzed by qPCR showed no difference between any of the treatments in Experiment 1 or 2. In both experiments, no treatment effects on the cecal microbiota were observed. However, proportional changes in the bacterial composition were observed after the C. jejuni challenge, suggesting that colonization affected the gut

Microbial symbionts range from mutualistic to commensal to antagonistic. While these roles are distinct in their outcome, they are also fluid in a changing environment. Here, we used the Lotus japonicus-Mesorhizobium japonicum symbiosis to investigate short-term and long-term shifts in population abundance using an effective, fast, and low-cost tracking methodology for M. japonicum. We use quantitative polymerase chain reaction (qPCR) to track previously generated signature-tagged M. japonicum mutants targeting the Tn5 transposon insertion and the flanking gene. We used a highly beneficial wild type and moderately beneficial and non-beneficial mutants of M. japonicum sp. nov. to demonstrate the specificity of these primers to estimate the relative abundance of each genotype within individual nodules and after serial transfers to new hosts. For the moderate and non-beneficial genotypes, qPCR allowed us to differentiate genotypes that are phenotypically indistinguishable and investigate host control with suboptimal symbionts. We consistently found the wild type increasing in the proportion of the population, but our data suggest a potential reproductive trade-off between the moderate and non-beneficial genotypes. The multi-generation framework we used, coupled with qPCR, can easily be scaled up to track dozens of M. japonicum mutants simultaneously. Moreover, these mutants can be used to explore M. japonicum genotype abundance in the presence of a complex soil community.

A voluntary upper respiratory biosurveillance program in the USA received 9740 nasal swab submissions during the years 2008-2021 from 333 veterinarians and veterinary clinics. The nasal swabs were submitted for qPCR testing for six common upper respiratory pathogens:equine influenza virus (EIV), equine herpesvirus-1 (EHV-1), equine herpesvirus-4 (EHV-4), Streptococcus equi subspecies equi (S. equi), equine rhinitis A virus (ERAV), and equine rhinitis B virus (ERBV). Additional testing was performed for equine gamma herpesvirus-2 (EHV-2) and equine gamma herpesvirus-5 (EHV-5) and the results are reported. Basic frequency statistics and multivariate logistic regression models were utilized to determine the associations between risk factors and EIV positivity. The EIV qPCR-positivity rate was 9.9%. Equids less than 9 years of age with a recent history of travel and seasonal occurrence in winter and spring were the most common population that were qPCR positive for EIV. This ongoing biosurveillance program emphasizes the need for molecular testing for pathogen identification, which is critical for decisions associated with therapeutics and biosecurity intervention for health management and vaccine evaluations and development.

Little information is presently available regarding the frequency of the silent shedders of respiratory viruses in healthy sport horses and their impact on environmental contamination. Therefore, the aim of this study was to investigate the detection frequency of selected respiratory pathogens in nasal secretions and environmental stall samples of sport horses attending a multi-week equestrian event during the summer months. Six out of fifteen tents were randomly selected for the study with approximately 20 horse/stall pairs being sampled on a weekly basis. Following weekly collection for a total of 11 weeks, all samples were tested for the presence of common respiratory pathogens (EIV, EHV-1, EHV-4, ERAV, ERBV, and Streptococcus equi ss equi (S. equi)) using qPCR. A total of 19/682 nasal swabs (2.8%) and 28/1288 environmental stall sponges (2.2%) tested qPCR-positive for common respiratory pathogens. ERBV was the most common respiratory virus (17 nasal swabs, 28 stall sponges) detected, followed by EHV-4 (1 nasal swab) and S. equi (1 nasal swab). EIV, EHV-1, EHV-4 and ERAV were not detected in any of the study horses or stalls. Only one horse and one stall tested qPCR-positive for ERBV on two consecutive weeks. All the other qPCR-positive sample results were related to individual time points. Furthermore, only one horse/stall pair tested qPCR-positive for ERBV at a single time point. The study results showed that in a selected population of sport horses attending a multi-week equestrian event in the summer, the frequency of the shedding of respiratory viruses was low and primarily restricted to ERBV with little evidence of active transmission and environmental contamination.

Recent studies have focused on how sickness behaviours, including lethargy, are coordinated in the brain in response to peripheral infections. Decreased hypocretin (orexin) signalling is associated with lethargy and previous research suggests that hypocretin signalling is downregulated during sickness. However, there are studies that find increases or no change in hypocretin signalling during sickness. It is further unknown whether hypocretin receptor expression changes during sickness. Using lipopolysaccharide (LPS) to induce sickness in female mice, we investigated how LPS-injection affects gene expression of hypocretin receptors and prepro-hypocretin as well as hypocretin-1 peptide concentrations in brain tissue. We found that hypocretin receptor 1 gene expression was downregulated during sickness in the lateral hypothalamus and ventral tegmental area, but not in the dorsal raphe nucleus or locus coeruleus. We found no changes in hypocretin receptor 2 expression. Using a gene expression calculation that accounts for primer efficiencies and multiple endogenous controls, we were unable to detect changes in prepro-hypocretin expression. Using radioimmunoassay, we found no change in hypocretin-1 peptide in rostral brain tissue. Our results indicate that hypocretin receptor expression can fluctuate during sickness, adding an additional level of complexity to understanding hypocretin signalling during sickness., Recent studies have focused on how sickness behaviours, including lethargy, are coordinated in the brain in response to peripheral infections. Decreased hypocretin (orexin) signalling is associated with lethargy and previous research suggests that hypocretin signalling is downregulated during sickness. However, there are studies that find increases or no change in hypocretin signalling during sickness. It is further unknown whether hypocretin receptor expression changes during sickness. Using lipopolysaccharide (LPS) to induce sickness in female mice, we investigated how LPS-injection affects gene expression of hypocretin receptors and prepro-hypocretin as well as hypocretin-1 peptide concentrations in brain tissue. We found that hypocretin receptor 1 gene expression was downregulated during sickness in the lateral hypothalamus and ventral tegmental area, but not in the dorsal raphe nucleus or locus coeruleus. We found no changes in hypocretin receptor 2 expression. Using a gene expression calculation that accounts for primer efficiencies and multiple endogenous controls, we were unable to detect changes in prepro-hypocretin expression. Using radioimmunoassay, we found no change in hypocretin-1 peptide in rostral brain tissue. Our results indicate that hypocretin receptor expression can fluctuate during sickness, adding an additional level of complexity to understanding hypocretin signalling during sickness.

Early detection and prompt response are key factors in the eradication of huanglongbing (HLB) in California. Currently, qPCR testing of leaf tissue guides the removal of infected trees. However, because of the uneven distribution of Candidatus Liberibacter asiaticus (CLas) in an infected tree and asymptomatic infection, selecting the best leaves to sample, from a mature tree with more than 200,000 estimated leaves, is a major hurdle for timely detection. The goal of this study was to address this issue by testing alternative tissues that might improve the CLas detection rate. Using two years of field data, old and young leaves, peduncle bark of fruit, and feeder roots were evaluated for the presence of CLas. Quadrant-peduncle (Q-P) tissue sampling consistently resulted in better CLas detection than any other tissue type. Q-P samples had a 30% higher qPCR positivity rate than quadrant-leaf (Q-L) samples. No significant seasonal patterns were observed. Roots and single peduncles had similar detection rates; both were higher than single leaves or Q-L samples. If symptoms were used to guide sampling, 30% of infected trees would have been missed. Taken together, these results suggest that Q-P tissue sampling is the optimal choice for improved CLas detection under California growing conditions.

Background Climate change has accelerated the occurrence and severity of heatwaves in the Mediterranean Sea and poses a significant threat to the octocoral species that form the foundation of marine animal forests (MAFs). As coral health intricately relies on the symbiotic relationships established between corals and microbial communities, our goal was to gain a deeper understanding of the role of bacteria in the observed tissue loss of key octocoral species following the unprecedented heatwaves in 2022. Results Using amplicon sequencing and taxon-specific qPCR analyses, we unexpectedly found that the absolute abundance of the major bacterial symbionts, Spirochaetaceae (C. rubrum) and Endozoicomonas (P. clavata), remained, in most cases, unchanged between colonies with 0% and 90% tissue loss. These results suggest that the impairment of coral health was not due to the loss of the main bacterial symbionts. However, we observed a significant increase in the total abundance of bacterial opportunists, including putative pathogens such as Vibrio, which was not evident when only their relative abundance was considered. In addition, there was no clear relation between bacterial symbiont loss and the intensity of thermal stress, suggesting that factors other than temperature may have influenced the differential response of octocoral microbiomes at different sampling sites. Conclusions Our results indicate that tissue loss in octocorals is not directly caused by the decline of the main bacterial symbionts but by the proliferation of opportunistic and pathogenic bacteria. Our findings thus underscore the significance of considering both relative and absolute quantification approaches when evaluating the impact of stressors on coral microbiome as the relative quantification does not accurately depict the actual changes in the microbiome. Consequently, this research enhances our comprehension of the intricate interplay between host organisms, their microbiomes, and environmenta

Le projet SECTOX porte sur l’étude de la contamination par les phycotoxines (PST et lipophiles dont DST) des huîtres creuses et des moules de la lagune de Thau ainsi que la dynamique temporelle et spatiale de cette contamination dans l’optique d’une gestion sectorisée de la lagune. Les campagnes de terrain ont été réalisées entre septembre 2019 et décembre 2020.La contamination des toxines lipophiles et des toxines hydrophiles (PST) a été suivie dans plusieurs matrices : coquillages et échantillonneurs passifs. Le suivi des espèces toxiques dans la colonne d’eau a été opéré selon plusieurs méthodes d’analyses et techniques d’échantillonnage. L’étude a mis en évidence que la contamination par les toxines lipophiles produites par une microalgue appartenant au genre toxique Dinophysis, impacte l’ensemble de la lagune de Thau sans qu’il soit possible de déterminer des dynamiques différentes entre les différents secteurs de la lagune. L’absence de prolifération du dinoflagellé Alexandrium pacificum (producteur de PST) sur la période suivie n’a pas permis de conclure sur l’existence d’une dynamique spatio-temporelle différente entre les secteurs de l’étang de Thau pour le risque de contamination par les PST. L’étude a aussi permis de tester de nouveaux outils (SPATT, échantillonneur passif pour les toxines) ainsi que la méthode de qPCR utilisant les outils de la biologie moléculaire pour l’identification et le dénombrement des espèces toxiques. Les résultats obtenus montrent que ces techniques sont prometteuses mais nécessitent une optimisation avant une utilisation en « routine » dans le volet sanitaire du réseau de surveillance REPHY.

The article processing charge was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) – 491192747 and the Open Access Publication Fund of Humboldt-Universität zu Berlin., Background: Quantifying infection intensity is a common goal in parasitological studies. We have previously shown that the amount of parasite DNA in faecal samples can be a biologically meaningful measure of infection intensity, even if it does not agree well with complementary counts of transmission stages (oocysts in the case of Coccidia). Parasite DNA can be quantified at relatively high throughput using quantitative polymerase chain reaction (qPCR), but amplification needs a high specificity and does not simultaneously distinguish between parasite species. Counting of amplified sequence variants (ASVs) from high-throughput marker gene sequencing using a relatively universal primer pair has the potential to distinguish between closely related co-infecting taxa and to uncover the community diversity, thus being both more specific and more open-ended. Methods: We here compare qPCR to the sequencing-based amplification using standard PCR and a microfluidics-based PCR to quantify the unicellular parasite Eimeria in experimentally infected mice. We use multiple amplicons to differentially quantify Eimeria spp. in a natural house mouse population. Results: We show that sequencing-based quantification has high accuracy. Using a combination of phylogenetic analysis and the co-occurrence network, we distinguish three Eimeria species in naturally infected mice based on multiple marker regions and genes. We investigate geographical and host-related effects on Eimeria spp. community composition and find, as expected, prevalence to be largely explained by sampling locality (farm). Controlling for this effect, the novel approach allowed us to find body condition of mice to be negatively associated with Eimeria spp. abundance. Conclusions: We conclude that amplicon sequencing provides the underused potential for species distinction and simultaneous quantification of parasites in faecal material. The method allowed us to detect a negative effect of Eimeria infection on the body, Peer Reviewed

Xanthomonas arboricola pv. corylina (Xac; formerly Xanthomonas campestris pv. corylina) is the causal agent of the bacterial blight of hazelnuts, a devastating disease of trees in plant nurseries and young orchards. Currently, there are no PCR assays to distinguish Xac from all other pathovars of X. arboricola. A comparative genomics approach with publicly available genomes of Xac was used to identify unique sequences, conserved across the genomes of the pathogen. We identified a 2,440 bp genomic region that was unique to Xac and designed identification and detection systems for conventional PCR, qPCR (SYBR® Green and TaqMan™), and loop-mediated isothermal amplification (LAMP). All PCR assays performed on genomic DNA isolated from eight X. arboricola pathovars and closely related bacterial species confirmed the specificity of designed primers. These new multi-platform molecular diagnostic tools may be used by plant clinics and researchers to detect and identify Xac in pure cultures and hazelnut tissues rapidly and accurately.

Arctic tundra fires have been increasing in extent, frequency and intensity and are likely impacting both soil nitrogen (N) and phosphorus (P) cycling and, thus, permafrost ecosystem functioning. However, little is known on the underlying microbial mechanisms, and different fire intensities were neglected so far. To better understand immediate influences of different fire intensities on the soil microbiome involved in nutrient cycling in permafrost-affected soil, we deployed experimental fires with low and high intensity on an Arctic tundra soil on Disko Island, Greenland. Soil sampling took place three days postfire and included an unburned control. Using quantitative real-time PCR, copy numbers of 16S and ITS as well as of 17 genes coding for functional microbial groups catalyzing major steps of N and P turnover were assessed. We show that fires change the abundance of microbial groups already after three days with fire intensity as key mediating factor. Specifically, low-intensity fire significantly enhanced the abundance of chiA mineralizers and ammonia-oxidizing archaea, while other groups were not affected. On the contrary, high-intensity fire decreased the abundance of chiA mineralizers and of microbes that fix dinitrogen, indicating a dampening effect on N cycling. Only high-intensity fires enhanced ammonium concentrations (by an order of magnitude). This can be explained by burned plant material and the absence of plant uptake, together with impaired further N processing. Fire with high intensity also decreased nirK-type denitrifiers. In contrast, after fire with low intensity there was a trend for a decreased nosZ: (nirK+nirS) ratio, indicating – together with increased nitrate concentrations – an enhanced potential for nitric oxide and nitrous oxide emissions. Concerning P transformation, only gcd was affected in the short term which is important for P solubilization. Changes in gene numbers consistently showed the same contrasting pattern of elevated

Begomoviruses (family Geminiviridae) cause serious diseases in many crop families. Since 2013, the Spanish isolate of tomato leaf curl New Delhi virus (ToLCNDV) has been a limiting factor for cucurbits production in the Mediterranean basin, forcing farmers to adapt new management and control techniques. Although it is well-known that begomoviruses are naturally transmitted by the whitefly Bemisia tabaci, the capacity of these viruses to be vertically transmitted through seeds remains controversial. Clarifying the potential ToLCNDV seed transmission is essential to understand the epidemiology of this threating-for-cucurbits virus and to design appropriate control strategies. We assessed ToLCNDV distribution in the leaves, flowers and seeds of the infected plants of susceptible Cucumis melo accessions and toleration to the infected genotypes of Cucurbita moschata by conventional and quantitative PCR. We analyzed whether the viral particle was transmitted to offspring. We also evaluated ToLCNDV presence in commercial seeds of cucurbits (zucchini (Cucurbita pepo), melon (C. melo), cucumber (Cucumis sativus) and watermelon (Citrullus lanatus)) and in their progenies. As the assayed seedlings remained symptomless, we increased the reliability and accuracy of detection in these samples by searching for replicative forms of ToLCNDV by combining Southern blot hybridization and rolling-circle amplification (RCA). However, integral genomic DNA was not identified in the plants of offspring. Although the seedborne nature of ToLCNDV was confirmed, our results do not support the transmission of this virus from contaminated seeds to progeny.