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Vet Parasitol,
2011]
The most challenging obstacles to testing products for their anthelmintic activity are: (1) establishing a suitable nematode in vitro assay that can evaluate potential product use against a parasitic nematode of interest and (2) preparation of extracts that can be redissolved in solvents that are miscible in the test medium and are at concentrations well tolerated by the nematode system used for screening. The use of parasitic nematodes as a screening system is hindered by the difficulty of keeping them alive for long periods outside their host and by the need to keep infected animals as sources of eggs or adults when needed. This method uses the free-living soil nematode Caenorhabditis elegans as a system to screen products for their potential anthelmintic effect against small ruminant gastrointestinal nematodes, including Haemonchus contortus. This modified method uses only liquid axenic medium, instead of agar plates inoculated with Escherichia coli, and two selective sieves to obtain adult nematodes. During screening, the use of either balanced salt solution (M-9) or distilled water resulted in averages of 99.7 (+/- 0.73)% and 96.36 (+/- 2.37)% motile adults, respectively. Adult worms tolerated DMSO, ethanol, methanol, and Tween 80 at 1% and 2%, while Labrasol (a bioenhancer with low toxicity to mammals) and Tween 20 were toxic to C. elegans at 1% and were avoided as solvents. The high availability, ease of culture, and rapid proliferation of C. elegans make it a useful screening system to test plant extracts and other phytochemical compounds to investigate their potential anthelmintic activity against parasitic nematodes.
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Vet Parasitol,
2013]
Although tannin-rich forages are known to increase protein uptake and to reduce gastrointestinal nematode infections in grazing ruminants, most published research involves forages with condensed tannins (CT), while published literature lacks information on the anthelmintic capacity, nutritional benefits, and antioxidant capacity of alternative forages containing hydrolyzable tannins (HT). We evaluated the anthelmintic activity and the antioxidant capacity of plant extracts containing either mostly CT, mostly HT, or both CT and HT. Extracts were prepared with 70% acetone, lyophilized, redissolved to doses ranging from 1.0mg/mL to 25mg/mL, and tested against adult Caenorhabditis elegans as a test model. The extract concentrations that killed 50% (LC(50)) or 90% (LC(90)) of the nematodes in 24h were determined and compared to the veterinary anthelmintic levamisole (8 mg/mL). Extracts were quantified for CT by the acid butanol assay, for HT (based on gallic acid and ellagic acid) by high-performance liquid chromatography (HPLC) and total phenolics, and for their antioxidant activity by the oxygen radical absorbance capacity (ORAC) assay. Extracts with mostly CT were Lespedeza cuneata, Salix X sepulcralis, and Robinia pseudoacacia. Extracts rich in HT were Acer rubrum, Rosa multiflora, and Quercus alba, while Rhus typhina had both HT and CT. The extracts with the lowest LC(50) and LC(90) concentrations, respectively, in the C. elegans assay were Q. alba (0.75 and 1.06 mg/mL), R. typhina collected in 2007 (0.65 and 2.74 mg/mL), A. rubrum (1.03 and 5.54 mg/mL), and R. multiflora (2.14 and 8.70 mg/mL). At the doses of 20 and 25mg/mL, HT-rich, or both CT- and HT-rich, extracts were significantly more lethal to adult C. elegans than extracts containing only CT. All extracts were high in antioxidant capacity, with ORAC values ranging from 1800 moles to 4651 moles of trolox equivalents/g, but ORAC did not correlate with anthelmintic activity. The total phenolics test had a positive and highly significant (r=0.826, p 0.01) correlation with total hydrolyzable tannins. Plants used in this research are naturalized to the Appalachian edaphoclimatic conditions, but occur in temperate climate areas worldwide. They represent a rich, renewable, and unexplored source of tannins and antioxidants for grazing ruminants, whereas conventional CT-rich forages, such as L. cuneata, may be hard to establish and adapt to areas with temperate climate. Due to their high in vitro anthelmintic activity, antioxidant capacity, and their adaptability to non-arable lands, Q. alba, R. typhina, A. rubrum, and R. multiflora have a high potential to improve the health of grazing animals and must have their anthelmintic effects confirmed in vivo in both sheep and goats.
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Int J Food Microbiol,
2016]
The current study explores the in vitro and in vivo antibiofilm efficacy of morin against a leading foodborne pathogen-Listeria monocytogenes (LM). Minimum inhibitory concentration (MIC) of morin against LM strains was found to be 100g/ml. The non-antibacterial effect of morin at its sub-MICs (6.25, 12.5 and 25g/ml) was determined through growth curve and XTT assay. Morin at its sub-MICs demonstrated a significant dose dependent inhibitory efficacy against LM biofilm formation which was also evidenced through light, confocal and scanning electron microscopic analyses. However, morin failed to disperse the mature biofilm of LM even at its MIC. Our data also revealed the anti-virulence efficacy of morin, as it significantly inhibited the production of hemolysin and motility of LM. Concentration-dependent susceptibility of morin treated LM cells to normal human serum was observed. In vivo studies revealed that morin extended the lifespan of LM infected Caenorhabditis elegans by about 85%. Furthermore, the non-toxic nature and in vivo anti-adherence efficacy of morin were also ascertained through C. elegans-LM infection model. Overall, the data of the current study identifies morin as a promising antibiofilm agent and its suitability to formulate protective strategies against biofilm associated infections caused by LM.
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Pathog Dis,
2016]
The current study was intentionally focused on cyclo(L-leucyl- L-prolyl) (CLP)-a cyclic dipeptide with myriad pharmaceutical significance, to explore its antivirulence efficacy against the predominant food-borne pathogen-Listeria monocytogenes (LM). Minimum inhibitory concentration (MIC) of CLP against LM ATCC 19111 was found to be 512 g mL(-1). CLP at sub-MICs (64,128, 256 g mL(-1)) demonstrated a profound non-bactericidal dose-dependent antibiofilm efficacy (on polystyrene and glass) against LM, which was further confirmed through confocal and scanning electron microscopic analysis (on stainless steel surface). In vitro bioassays divulged the phenomenal inhibitory efficacy of CLP towards various virulence traits of LM, specifically its overwhelming suppression of swimming and swarming motility. Data of in vivo assay using Caenorhabditis elegans signified that the plausible mechanism of CLP could be by impeding the pathogen's initial adhesion and thereby attenuating the biofilm assemblage and its associated virulence. This was further confirmed by significant decrease in exopolymeric substance, auto-aggregation, hydrophobicity index and extracellular DNA (eDNA) of the CLP treated-LM cells. Collectively, the current study unveils the antivirulence efficacy of CLP against the Gram-positive food borne-pathogen and the strain Bacillus amyloliquifaciens augurs well to be a promising probiotic in controlling infections associated with LM.
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Nat Methods,
2023]
Our understanding of nerve regeneration can be enhanced by delineating its underlying molecular activities at single-neuron resolution in model organisms such as Caenorhabditis elegans. Existing cell isolation techniques cannot isolate neurons with specific regeneration phenotypes from C. elegans. We present femtosecond laser microdissection (fs-LM), a single-cell isolation method that dissects specific cells directly from living tissue by leveraging the micrometer-scale precision of fs-laser ablation. We show that fs-LM facilitates sensitive and specific gene expression profiling by single-cell RNA sequencing (scRNA-seq), while mitigating the stress-related transcriptional artifacts induced by tissue dissociation. scRNA-seq of fs-LM isolated regenerating neurons revealed transcriptional programs that are correlated with either successful or failed regeneration in wild-type and
dlk-1 (0) animals, respectively. This method also allowed studying heterogeneity displayed by the same type of neuron and found gene modules with expression patterns correlated with axon regrowth rate. Our results establish fs-LM as a spatially resolved single-cell isolation method for phenotype-to-genotype mapping.
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Methods Cell Biol,
2017]
Correlative light and electron microscopy (CLEM) is a powerful tool to perform ultrastructural analysis of targeted tissues or cells. The large field of view of the light microscope (LM) enables quick and efficient surveys of the whole specimen. It is also compatible with live imaging, giving access to functional assays. CLEM protocols take advantage of the features to efficiently retrace the position of targeted sites when switching from one modality to the other. They more often rely on anatomical cues that are visible both by light and electron microscopy. We present here a simple workflow where multicellular specimens are embedded in minimal amounts of resin, exposing their surface topology that can be imaged by scanning electron microscopy (SEM). LM and SEM both benefit from a large field of view that can cover whole model organisms. As a result, targeting specific anatomic locations by focused ion beam-SEM (FIB-SEM) tomography becomes straightforward. We illustrate this application on three different model organisms, used in our laboratory: the zebrafish embryo Danio rerio, the marine worm Platynereis dumerilii, and the dauer larva of the nematode Caenorhabditis elegans. Here we focus on the experimental steps to reduce the amount of resin covering the samples and to image the specimens inside an FIB-SEM. We expect this approach to have widespread applications for volume electron microscopy on multiple model organisms.
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Methods Cell Biol,
2012]
The rationale of correlative light and electron microscopy (CLEM) is to collect data on different information levels--ideally from an identical area on the same sample--with the aim of combining datasets at different levels of resolution to achieve a more holistic view of the hierarchical structural organization of cells and tissues. Modern three-dimensional (3D) imaging techniques in light and electron microscopy opened up new possibilities to expand morphological studies into the third dimension at the nanometer scale and over various volume dimensions. Here, we present two alternative approaches to correlate 3D light microscopy (LM) data with scanning electron microscopy (SEM) volume data. An adapted sample preparation method based on high-pressure freezing for structure preservation, followed by freeze-substitution for multimodal en-bloc imaging or serial-section imaging is described. The advantages and potential applications are exemplarily shown on various biological samples, such as cells, individual organisms, human tissue, as well as plant tissue. The two CLEM approaches presented here are per se not mutually exclusive, but have their distinct advantages. Confocal laser scanning microscopy (CLSM) and focused ion beam-SEM (FIB-SEM) is most suitable for targeted 3D correlation of small volumes, whereas serial-section LM and SEM imaging has its strength in large-area or -volume screening and correlation. The second method can be combined with immunocytochemical methods. Both methods, however, have the potential to extract statistically relevant data of structural details for systems biology.
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Costa-Junior LM, Tamboura HH, Bayala B, Kabore A, Louvandini H, Belem AMG, Katiki LM, Zabre G, Hoste H, Abdalla AL, Niderkorn V
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Parasite,
2017]
Gastrointestinal nematodes are a major threat to small ruminant rearing in the Sahel area, where farmers traditionally use bioactive plants to control these worms, including Acacia nilotica and Acacia raddiana. The main aim of this study was to screen the potential anthelmintic properties of aqueous and acetone extracts of leaves of these two plants based on three in vitro assays: (1) the egg hatch inhibition assay (EHA); (2) the larvae exsheathment inhibition assay (LEIA) using Haemonchus contortus as a model; and (3) an adult mortality test (AMT) applied on Caenorhabditis elegans. For the EHA, only A. raddiana was effective with IC50 = 1.58 mg/mL for aqueous extract, and IC50 = 0.58 mg/mL for acetonic extract. For the LEIA, all extracts inhibited the exsheathment of larvae compared to the controls, and the aqueous extract of A. nilotica was more larvicidal with IC50 = 0.195 mg/mL. In general, all responses to the substances were dose-dependent and were significantly different from the control group (p < 0.05). For the AMT, the extracts of the two Acacia species were effective but A. raddiana showed greater efficacy with 100% mortality at 2.5 mg/mL and LC50 = 0.84 mg/mL (acetonic extract). The addition of polyvinyl polypyrrolidone (PVPP) to the extracts suggested that tannins were responsible for blocking egg eclosion and inducing adult mortality but were not responsible for exsheathment inhibition. These results suggest that the leaves of these Acacia species possess ovicidal and larvicidal activities in vitro against H. contortus, and adulticidal effects against C. elegans.