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The digestion of biogenic organic matter is an essential step of sample preparation within microplastic analyses. Organic residues hamper the separation of polymer particles especially within density separation or polymer identification via spectroscopic and staining methods. Therefore, a concise literature survey has been undertaken to identify the most commonly applied digestion protocols with a special focus on water and sediments samples. The selected protocols comprise different solutions, concentrations, and reaction temperatures. Within this study we tested acids (nitric acid and hydrochloric acid), bases (sodium hydroxide and potassium hydroxide), and oxidizing agents [hydrogen peroxide, sodium hypochlorite and Fenton's reagent (hydrogen peroxide 30% in combination with iron(II)sulfate 0.27%)] at different concentrations, temperature levels, and reaction times on their efficiency of biogenic organic matter destruction and the resistance of different synthetic polymers against the applied digestion protocols. Tests were carried out in three parallels on organic material (soft tissue—leaves, hard tissue—branches, and calcareous material—shells) and six polymers (low-density polyethylene, high-density polyethylene, polypropylene, polyamide, polystyrene, and polyethylene terephthalate) in two size categories. Before and after the application of different digestion protocols, the material was weighed in order to determine the degree of digestion efficiency and polymer resistance, respectively. The efficiency of organic matter destruction is highly variable. Calcareous shells showed no to very low reaction to oxidizing agents and bases, but were efficiently dissolved with both tested acids at all concentrations and at all temperatures. Soft and hard tissue were most efficiently destroyed by sodium hypochlorite. However, the other reagents can also have good effects, especially by increasing the temperature to 40–50°C. The additional temperature increase to 60–70°C showed a further but less effective improvement, compared to the initial temperature increase. The resistance of tested polymer types can be rated as good except for polyamide and polyethylene terephthalate. Increasing the concentrations and temperatures, however, results in accelerated degradation of all polymers. This is most evident for polyamide and polyethylene terephthalate, which show losses in weight between 15 and 100% when the digestion temperature is increased. This effect is most pronounced for polyamide in the presence of acids and for polyethylene terephthalate digested with bases. As a concluding recommendation the selection of the appropriate digestion method should be specifically tested within initial pre-tests to account for the specific composition of the sample matrix and the project objectives.

A variety of methods concerning the identification of microplastics in environmental samples exist. While visual identification is often used, implemented easily, and cost-efficient but implying biased results, spectroscopic or chromatographic approaches are reliable but time-consuming and need specific equipment. Nile red staining is an available alternative and complement method for identifying microplastics. In this study, Nile red staining and subsequent photographing in a UV light photobox was tested on its reliability and feasibility. The approach was compared with a second identification process using again staining but a fluorescence microscope. Selected identified microplastic particles were analyzed by μ-Raman spectroscopy to prove their polymeric origin. The results show that the presented approach is faster compared with the use of a fluorescence microscope or μ-Raman spectroscopy. Furthermore, it is cost-effective as well as accurate for large microplastics > 0.63 mm and, therefore, may be applied when large sample volumes need to be analyzed. Graphical abstract

Food borne trematodes (FBTs) are an assemblage of platyhelminth parasites transmitted through the food chain, four of which are recognized as neglected tropical diseases (NTDs). Fascioliasis stands out among the other NTDs due to its broad and significant impact on both human and animal health, as Fasciola sp., are also considered major pathogens of domesticated ruminants. Here we present a reference genome sequence of the common liver fluke, Fasciola hepatica isolated from sheep, complementing previously reported isolate from cattle. A total of 14,642 genes were predicted from the 1.14 GB genome of the liver fluke. Comparative genomics indicated that F. hepatica Oregon and related food-borne trematodes are metabolically less constrained than schistosomes and cestodes, taking advantage of the richer millieux offered by the hepatobiliary organs. Protease families differentially expanded between diverse trematodes may facilitate migration and survival within the heterogeneous environments and niches within the mammalian host. Surprisingly, the sequencing of Oregon and Uruguay F. hepatica isolates led to the first discovery of an endobacteria in this species. Two contigs from the F. hepatica Oregon assembly were joined to complete the 859,205 bp genome of a novel Neorickettsia endobacterium (nFh) closely related to the etiological agents of human Sennetsu and Potomac horse fevers. Immunohistochemical studies targeting a Neorickettsia surface protein found nFh in specific organs and tissues of the adult trematode including the female reproductive tract, eggs, the Mehlis' gland, seminal vesicle, and oral suckers, suggesting putative routes for fluke-to-fluke and fluke-to-host transmission. The genomes of F. hepatica and nFh will serve as a resource for further exploration of the biology of F. hepatica, and specifically its newly discovered trans-kingdom interaction with nFh and the impact of both species on disease in ruminants and humans.

Brugia malayi is the most common cause of lymphatic filariasis (LF) in Indonesia. A zoophilic ecotype that infects both humans and animals occur in Belitung District in Indonesia. The district received five annual rounds of mass drug administration (MDA) between 2006 and 2010 and passed three transmission assessment surveys (TAS) in subsequent years. However, a survey in five villages in 2021 showed a microfilaria (Mf) prevalence of 2.1% in humans. The reappearance of B. malayi infection in humans may be due to reintroduction from animal reservoirs. The goal of this study was to determine B. malayi prevalence in potential reservoir hosts and to improve the identification of filarial Mf found in animals. Venous blood was collected from 291 cats, 41 dogs, and 163 crab-eating macaques (Macaca fascicularis) from areas with and without human B. malayi infection. B. malayi Mf were detected by microscopy in 1.4%, 7.3% and 13.5% of the samples, respectively. The geometric mean Mf density varied from 133 Mf/mL(dogs) to 255 Mf/mL (macaques). While Brugia Mf were easily differentiated from Dirofilaria Mf by microscopy, the morphological differentiation between B. malayi and B. pahangi was not reliable. qPCR detected B. malayi DNA in blood from 4.1% of cats, 2.4% dogs, and 13.5% macaques. In addition, infections or co-infection with B. pahangi (cats, dogs) or D. immitis (dogs) were detected. A novel Dirofilaria species was morphologically identified in 20.3% of macaques. Microscopy was less accurate for detection and species identification of Mf than qPCR. The presence of B. malayi Mf in animals represents a challenge for the elimination of LF in some areas in Indonesia. More research is needed to better understand B. malayi transmission between animals and humans in endemic areas like Belitung where routine MDA may not be sufficient to eliminate LF.

Background The human intestine and its microbiota is the most common infection site for soil-transmitted helminths (STHs), which affect the well-being of ~ 1.5 billion people worldwide. The complex cross-kingdom interactions are not well understood. Results A cross-sectional analysis identified conserved microbial signatures positively or negatively associated with STH infections across Liberia and Indonesia, and longitudinal samples analysis from a double-blind randomized trial showed that the gut microbiota responds to deworming but does not transition closer to the uninfected state. The microbiomes of individuals able to self-clear the infection had more alike microbiome assemblages compared to individuals who remained infected. One bacterial taxon ( Lachnospiracae ) was negatively associated with infection in both countries, and 12 bacterial taxa were significantly associated with STH infection in both countries, including Olsenella (associated with reduced gut inflammation), which also significantly reduced in abundance following clearance of infection. Microbial community gene abundances were also affected by deworming. Functional categories identified as associated with STH infection included arachidonic acid metabolism; arachidonic acid is the precursor for pro-inflammatory leukotrienes that threaten helminth survival, and our findings suggest that some modulation of arachidonic acid activity in the STH-infected gut may occur through the increase of arachidonic acid metabolizing bacteria. Conclusions For the first time, we identify specific members of the gut microbiome that discriminate between moderately/heavily STH-infected and non-infected states across very diverse geographical regions using two different statistical methods. We also identify microbiome-encoded biological functions associated with the STH infections, which are associated potentially with STH survival strategies, and changes in the host environment. These results provide a novel insight of the cross-kingdom interactions in the human gut ecosystem by unlocking the microbiome assemblages at taxonomic, genetic, and functional levels so that advances towards key mechanistic studies can be made.

Cedar henipavirus (CedV), which was isolated from the urine of pteropodid bats in Australia, belongs to the genus Henipavirus in the family of Paramyxoviridae. It is closely related to the Hendra virus (HeV) and Nipah virus (NiV), which have been classified at the highest biosafety level (BSL4) due to their high pathogenicity for humans. Meanwhile, CedV is apathogenic for humans and animals. As such, it is often used as a model virus for the highly pathogenic henipaviruses HeV and NiV. In this study, we challenged eight Rousettus aegyptiacus fruit bats of different age groups with CedV in order to assess their age-dependent susceptibility to a CedV infection. Upon intranasal inoculation, none of the animals developed clinical signs, and only trace amounts of viral RNA were detectable at 2 days post-inoculation in the upper respiratory tract and the kidney as well as in oral and anal swab samples. Continuous monitoring of the body temperature and locomotion activity of four animals, however, indicated minor alterations in the challenged animals, which would have remained unnoticed otherwise.

We studied the localization of 6-phosphogluconate dehydrogenase (PGD) isoforms of Arabidopsis (Arabidopsis thaliana). Similar polypeptide lengths of PGD1, PGD2, and PGD3 obscured which isoform may represent the cytosolic and/or plastidic enzyme plus whether PGD2 with a peroxisomal targeting motif also might target plastids. Reporter-fusion analyses in protoplasts revealed that, with a free N terminus, PGD1 and PGD3 accumulate in the cytosol and chloroplasts, whereas PGD2 remains in the cytosol. Mutagenesis of a conserved second ATG enhanced the plastidic localization of PGD1 and PGD3 but not PGD2. Amino-terminal deletions of PGD2 fusions with a free C terminus resulted in peroxisomal import after dimerization, and PGD2 could be immunodetected in purified peroxisomes. Repeated selfing of pgd2 transfer (T-)DNA alleles yielded no homozygous mutants, although siliques and seeds of heterozygous plants developed normally. Detailed analyses of the C-terminally truncated PGD2-1 protein showed that peroxisomal import and catalytic activity are abolished. Reciprocal backcrosses of pgd2-1 suggested that missing PGD activity in peroxisomes primarily affects the male gametophyte. Tetrad analyses in the quartet1-2 background revealed that pgd2-1 pollen is vital and in vitro germination normal, but pollen tube growth inside stylar tissues appeared less directed. Mutual gametophytic sterility was overcome by complementation with a genomic construct but not with a version lacking the first ATG. These analyses showed that peroxisomal PGD2 activity is required for guided growth of the male gametophytes and pollen tube-ovule interaction. Our report finally demonstrates an essential role of oxidative pentose-phosphate pathway reactions in peroxisomes, likely needed to sustain critical levels of nitric oxide and/or jasmonic acid, whose biosynthesis both depend on NADPH provision.

Onchocerca volvulus is the agent of onchocerciasis (river blindness) and targeted by WHO for elimination though mass drug administration with ivermectin. A small percentage of adult female worms develop pleomorphic neoplasms (PN) which occur more frequently after ivermectin treatment. Worms with PN have a lower life expectancy and improved understanding of proteins expressed in PN and their impact on different tissues could help elucidate the mechanisms of macrofilaricidal activity of ivermectin. Within paraffin embedded nodules removed after ivermectin treatment, we detected 24 (5.6%) O. volvulus females with PN. To assess the protein inventory of the PN and identify proteins potentially linked with tumor development, we used laser capture microdissection and highly sensitive mass spectrometry analysis. Three female worms were used to compare the protein profiles of three tissue types (body wall, uterus, and intestine) to the PN, and then to healthy female worms without PN. The healthy females showed all normal embryogenesis. In PN worms, 151 proteins were detected in the body wall, 215 proteins in the intestine, 47 proteins in the uterus and 1,577 proteins in the PN. Only the uterus of one PN female with some stretched intrauterine microfilariae had an elevated number of proteins (601) detectable, while in the uteri of the healthy females 1,710 proteins were detected. Even in tissues that were not directly affected by PN (intestine, body wall), fewer proteins were detected compared to the corresponding tissue of the healthy controls. Immunolocalization of calcium binding protein OvDig-1 (OVOC8391), which was identified through mass spectrometry as one of the proteins with the highest spectral counts in the PN tissue triplicates, allowed us to confirm the results using an independent method. In conclusion we identified proteins that are potentially linked to the development of PN, and systemic dysregulation of protein expression may contribute to worm mortality.

Genomic analysis of parasites can deepen our understanding of their transmission, population structure, and important biological characteristics. Onchocerciasis (river blindness), caused by the parasitic nematode Onchocerca volvulus , involves adult worms residing in subcutaneous nodules that produce larval-stage microfilariae (mf), which are routinely detected in the skin for diagnosis. Whole-genome studies of mf are limited; most analyses have focused on the mitochondrial genome. We conducted a genome-wide analysis with 94% median nuclear genome coverage, analyzing 171, 37, and 98 mf from 16, 3, and 5 individuals from Ghana, Liberia, and the Democratic Republic of Congo, respectively. These data were used to investigate population differentiation, estimate the number of reproductive adult worms, and analyze genetic variation across chromosomes. Population genetic analyses across hosts and countries showed that nuclear genome diversity can reveal fine-scale genetic structure, even between geographically close countries, providing more resolution than mitochondrial haplotype data. By reconstructing maternal and paternal sibships, we estimated the number of reproductively active adult filariae. Comparisons between adult worm estimates from genetic data and nodule observations showed that genetics-based estimates were higher or equal to observed worm counts in 8 out of 9 hosts for female worms and 7 out of 9 hosts for male worms. Our analysis also revealed lower-than-expected X chromosome diversity, consistent with neo-X chromosome fusions in filarial species. This study represents an important step in using nuclear genome data from mf to support onchocerciasis elimination efforts and in developing genetic tools that could inform mass drug administration programs.