Worm Viability Research Articles

Methanolic Extract of Rhizophora mangle (Rhizophoraceae) Leaves: Phytochemical Characterization and Anthelmintic Evaluation against Schistosoma mansoni.

Rhizophora mangle is commonly used in traditional medicine to treat infections, reduce inflammation, and promote healing. This study aimed to analyze the phytochemical profile of the methanolic extract of R. mangle leaves (MELRm) and evaluate its in vitro schistosomicidal activity against Schistosoma mansoni as well as its cytotoxicity. Plant material was collected in Itamaracá City, Pernambuco, Brazil. The extract was analyzed using UV/Vis spectrophotometry and high-performance liquid chromatography (HPLC). The motility, mortality, and cell viability of adult worms were assessed in a schistosomicidal assay, while cytotoxicity was evaluated through a colorimetric assay with MTT on RAW 264.7 cells. The primary compounds identified in MELRm were phenolic compounds. In the schistosomicidal assay, all concentrations of MELRs induced changes in the motility of adult worms. At a concentration of 400 μg/mL, MELRs resulted in 56.25% mortality after 72 h of incubation. After 120 h, mortality rates of 75%, 62.5%, and 50% were observed at MELRm concentrations of 400, 200, and 100 μg/mL, respectively. No eggs were detected at any MELRm concentration. MELRs did not show cytotoxicity towards RAW 264.7 cells at the concentrations tested. These results indicate that MELRs demonstrate schistosomicidal activity in vitro, suggesting they are promising candidates for in vivo studies.

Human Placental Schistosomiasis-A Systematic Review of the Literature.

Schistosome egg deposition in pregnant women may affect the placenta of infected mothers and cause placental schistosomiasis (PS). Histopathological examination of placental tissue is an inadequate detection method due to low sensitivity. So far, there has not been any systematic review on PS. We conducted a systematic literature search on PubMed, EMBASE, and Medline and included all publications that reported microscopically confirmed cases of PS, as well as the relevant secondary literature found in the citations of the primarily included publications. Out of 113 abstracts screened we found a total of 8 publications describing PS with a total of 92 cases describing egg deposition of dead and/or viable eggs and worms of S. haematobium and S. mansoni in placental tissue. One cross-sectional study investigating the prevalence of PS and its association with adverse birth outcomes, found 22% of placentas to be infested using a maceration technique but only <1% using histologic examination. Additionally, no direct link to deleterious pregnancy outcomes could be shown. PS is a highly unattended and underdiagnosed condition in endemic populations, due to a lack of awareness as well as low sensitivity of histopathological examinations. However, PS may play an important role in mediating or reinforcing adverse birth outcomes (ABO) such as fetal growth restriction (FGR) in maternal schistosomiasis, possibly by placental inflammation.

Differential susceptibility of Onchocerca ochengi adult male worms to flubendazole in gerbils and hamsters.

Onchocerciasis is a devastating skin and eye disease that afflicts about 21 million people, most of whom live in sub-Saharan Africa. Its control with the microfilaricidal drug ivermectin is limited, thus necessitating the development of preclinical animal models to aid in the discovery of a macrofilaricide. Previously, we found that Onchocerca ochengi (the closest relative of the human O. volvulus) worm masses survive better in hamsters than in gerbils. The aim of this study was to compare the survival of O. ochengi adult male worms and their susceptibility to flubendazole (FBZ, a macrofilaricide) in gerbils and hamsters. The animals were intraperitoneally implanted with O. ochengi male worms, treated with FBZ, and sacrificed 35days post-implantation. Unlike gerbils which had some worms moving freely in the peritoneum and some in newly formed nodules (neo-nodules), all the worms in the hamsters were found in neo-nodules. FBZ significantly decreased worm burden, motility, and viability in gerbils whereas it had no significant effect in hamsters. These results highlight a major difference in how O. ochengi adult male worms are sustained and affected by FBZ in gerbils compared to hamsters. Understanding the difference between these two models is important in the development of effective macrofilaricides for onchocerciasis.

Exploring the Impact of Onobrychis cornuta and Veratrum lobelianum Extracts on C. elegans: Implications for MAPK Modulation, Germline Development, and Antitumor Properties.

In an era of increasing interest in the potential health benefits of medicinal foods, the need to assess their safety and potential toxicity remains a critical concern. While these natural remedies have garnered substantial attention for their therapeutic potential, a comprehensive understanding of their effects on living organisms is essential. We examined 316 herbal extracts to determine their potential nematocidal attributes in Caenorhabditis elegans. Approximately 16% of these extracts exhibited the capacity to induce diminished survival rates and larval arrest, establishing a correlation between larval arrest and overall worm viability. Certain extracts led to an unexpected increase in male nematodes, accompanied by a discernible reduction in DAPI-stained bivalent structures and perturbed meiotic advancement, thereby disrupting the conventional developmental processes. Notably, Onobrychis cornuta and Veratrum lobelianum extracts activated a DNA damage checkpoint response via the ATM/ATR and CHK-1 pathways, thus hindering germline development. Our LC-MS analysis revealed jervine in V. lobelianum and nine antitumor compounds in O. cornuta. Interestingly, linoleic acid replicated phenotypes induced by O. cornuta exposure, including an increased level of pCHK-1 foci, apoptosis, and the MAPK pathway. Mutants in the MAPK pathway mitigated the decline in worm survival, underscoring its importance in promoting worm viability. This study reveals complex interactions between herbal extracts and C. elegans processes, shedding light on potential antitumor effects and mechanisms. The findings provide insights into the complex landscape of herbal medicine's impact on a model organism, offering implications for broader applications.

Encapsulation of Caenorhabditis elegans in Water-in-Water Microdroplets to Study the Worm Viability: Alternative Avenue to Manipulate Microdroplet Environment.

Due to the great biocompatibility of the aqueous two phase system (ATPS), biological cells have been widely encapsulated in ATPS microdroplets (diameter < 50 μm). However, the immobilization of relatively large multicellular organisms such as Caenorhabditis elegans in ATPS droplets remains challenging as the spontaneous generation of droplets greater than 200 μm is difficult without external perturbations. In this study, we utilize a microneedle-assisted coflow microfludic channel to passively form ATPS microdroplets larger than 200 μm and successfully entrap C. elegans in the microdroplets. We monitor the worm viability and its temporal stroke frequency up to 6 h. We study the effects of dextran (DEX)-to-polyethylene glycol (PEG) flow ratios and worm concentration on the droplet diameter, worm encapsulation efficiency, and the number of droplets containing individual worms. Larger ATPS microdroplets (>200 μm) form in the ranges of capillary number (Ca) between 0.020 to 0.20 and Weber number (We) between 10-5 and 10-3. An ATPS with the encapsulation ability and biocompatibility can offer an alternative immobilization tool for multicellular organisms to existing platforms such as water/oil droplets.

In vitro Anthelmintic Activity of Croton tiglium Seeds Extract on Haemonchus contortus

Background: Haemonchus contortus is a blood sucking parasite whose habitat is the abomasum of ruminants, which poses a significant threat to the health and production of sheep and goats in warm temperate and tropical regions. H. contortus leads to anemia, hypoproteinemia, reduced exercise tolerance and edema subcutaneous tissue that can result in the death of animals. In acute infection, animals suddenly become anemic and many are found dead. Even in the chronic phase, the condition is accompanied by a mild but persistent parasitic burden and is characterized by weight loss, widespread malaise and, in some individuals, anemia. The objective of this study was to identify the anthelmintic activity of Croton tiglium seeds extract against Haemonchus contortus and comparison with some drugs used against internal and external parasites. Methods: A study was performed to evaluate the anthelmintic activity of the methanolic extract of Croton tiglium seeds against Haemonchus contortus using an adult worm motility test. Six graduated concentrations of extract (25, 50, 75, 100, 125 and 150 mg/ml), were tested at different periods and changes over time in the viability of worms were registered for 2, 4 and 6 hr. Normal RPMI-1640 medium and ivermectin 0.1% were used as negative and positive control, respectively. Also, a histological study was conducted on the the integumentary tissue of the H. contortus and the changes that occur in it were observed. Result: At 2 and 4 h post treatment, the concentrations of C. tiglium seeds extract (100, 125, and 150 mg/ml) have caused significantly higher mortality compared to the other concentrations, while, similar to ivermectin that caused death for nematodes within 6 h. The lower concentration (25 mg/ml) was significantly more lethal than the negative control (RPMI-1640 medium) at 2, 4 and 6 h of exposure. Each concentrations damages the cuticle and muscles of H. contortus. The increase in concentration is proportional to the increase in damage to the integumentary. The present study indicated that all concentrations of methanolic extract of C. tiglium seeds produced anthelmintic activity.

In vitro anthelminthic activity and ultrastructural analysis of barbatic acid against schistosomulae and juvenile worms of Schistosoma mansoni.

Schistosomiasis affects about 260 million people worldwide and the search for new schistosomicidal compounds is urgent. In this study we evaluated the in vitro effect of barbatic acidagainst schistosomulae and young worms of Schistosoma mansoni. The barbatic acid was evaluated through the bioassay of motility and mortality, cellular viability and ultrastructural analysis of juvenile stages through Scanning Electron Microscopy. Barbatic acid showed a schistosomicidal effect against schistosomulae and young worms of S. mansoni after 3 h of exposure. At the end of 24 h, barbatic acid showed 100%, 89.5%, 52% and 28.5% of lethality for schistosomulae at the concentrations of 200, 100, 50 and 25 µM, respectively. For young worms, barbatic acid showed 100% and 31.7% of lethality at the concentrations of 200 and 100 µM, respectively. Motility changes were observed at all sublethal concentrations. There was a significant reduction in the viability of young worms after exposure to barbatic acid at 50, 100 and 200 µM. Extensive damage to the schistosomulae and young worm's tegument, was observed from 50 μM. This report provides data showing the schistosomicidal effect of barbatic acid on schistosomulae and young worms of S. mansoni, causing death, motility changes and ultrastructural damage to worms.

A randomized, open-label study of the tolerability and efficacy of one or three daily doses of ivermectin plus diethylcarbamazine and albendazole (IDA) versus one dose of ivermectin plus albendazole (IA) for treatment of onchocerciasis.

Onchocerciasis ("river blindness") has been targeted for elimination. New treatments that kill or permanently sterilize female worms could accelerate this process. Prior studies have shown that triple drug treatment with ivermectin plus diethylcarbamazine and albendazole (IDA) leads to prolonged clearance of microfilaremia in persons with lymphatic filariasis. We now report results from a randomized clinical trial that compared the tolerability and efficacy of IDA vs. a comparator treatment (ivermectin plus albendazole, IA) in persons with onchocerciasis. The study was performed in the Volta region of Ghana. Persons with microfiladermia and palpable subcutaneous nodules were pre-treated with two oral doses of ivermectin (150 μg/kg) separated by at least 6 months prior to treatment with either a single oral dose of ivermectin 150 μg/kg plus albendazole 400 mg (IA), a single oral dose of IDA (IDA1, IA plus diethylcarbamazine (DEC. 6 mg/kg) or three consecutive daily doses of IDA (IDA3). These treatments were tolerated equally well. While adverse events were common (approximately 30% overall), no severe or serious treatment-emergent adverse events were observed. Skin microfilariae were absent or present with very low densities after all three treatments through 18 months, at which time nodules were excised for histological assessment. Nodule histology was evaluated by two independent assessors who were masked regarding participant infection status or treatment assignment. Significantly lower percentages of female worms were alive and fertile in nodules recovered from study participants after IDA1 (40/261, 15.3%) and IDA3 (34/281, 12.1%) than after IA (41/180, 22.8%). This corresponds to a 40% reduction in the percentage of female worms that were alive and fertile after IDA treatments relative to results observed after the IA comparator treatment (P = 0.004). Percentages of female worms that were alive (a secondary outcome of the study) were also lower after IDA treatments (301/574, 52.4%) than after IA (127/198, 64.1%) (P = 0.004). Importantly, some comparisons (including the reduced % of fertile female worms after IDA1 vs IA treatment, which was the primary endpoint for the study) were not statistically significant when results were adjusted for intraclass correlation of worm fertility and viability for worms recovered from individual study participants. Results from this pilot study suggest that IDA was well tolerated after ivermectin pretreatment. They also suggest that IDA was more effective than the comparator treatment IA for killing or sterilizing female O. volvulus worms. No other short-course oral treatment for onchocerciasis has been demonstrated to have macrofilaricidal activity. However, this first study was too small to provide conclusive results. Therefore, additional studies will be needed to confirm these promising findings. The study is registered at Cinicaltrials.gov under the number NCT04188301.

In vitro Anthelmintic Activity of Vitis vinifera Leaf Extract on Dicrocoelium dendriticum

Background: Helminth parasites of ruminants are a major factor limiting animal production in many parts of the world. Dicrocoelium dendriticum, causes liver fluke disease in ruminants and is of zoonotic and economic importance. D. dendriticum lives in the adult stage, in the bile ducts and gallbladders of cattle, sheep, goats and pigs. Dicrocoeliasis causes severe pathological changes in the liver and bile system such as abscesses, granulomas and fibrosis. The objective of this study was to identify the anthelmintic activity of Vitis vinifera leaf extract against D. dendriticum and comparison with some drugs used against internal parasites. Methods: A study was performed to evaluate the anthelmintic activity of the methanolic extract of V. vinifera leaf against D. dendriticum using an adult worm motility test. Six graduated concentrations of extract (25, 50, 75, 100, a125 and 150 mg/ml), were tested at different periods and changes over time in the viability of worms were registered for 2, 4 and 6 hr. Normal RPMI-1640 medium and Albendazole were used as negative and positive control, respectively. Result: A significant elevation in mean inhibition of adult worm motility beginning 2 h post-exposure with 150 mg/ml of V. vinifera leaf extract and Albendazole. After 4 h of the exposure time, Albendazole and concentrations of 125 and 150 mg/ml of the extract resulted in significantly higher inhibited motility compared to the remaining concentrations below 125 mg/ml. The lower concentration (25 mg/ml) was significantly more lethal than the negative control (RPMI-1640 medium) at 6 h of exposure. Within 6 h after exposure, concentrations of 100, 125 and 150 mg/ml of V. vinifera leaf extract were more effective against adult worms than the remaining concentrations below 100 mg/ml. As the concentration and duration of exposure increased, the mortality of adult worms and also increased death for all parasites (P less than 0.05). Each concentration damages the tegument and muscles of D. dendriticum. The increase in concentration is proportional to the increase in damage to the integument. The results of this investigation demonstrated the anthelmintic action of V. vinifera leaf extract.

Impact of temperature variations in worm containers and nutrition amount on silk glands and silk productivity

During the feeding of mulberry silkworms, when the temperature in the larvae fluctuates sharply and the amount of nutrients fed is below or above the norm, the metabolism and physiological processes in the body are disrupted. As a result, the silk cocoon water balance, respiration, digestion and absorption of nutrients are impaired, the activity of the silk glands is impaired, creating conditions for disease-causing microorganisms. In this case, the growth of silkworms by 4 times less when the worms are fed with 50% of the norm, reducing the amount of silk in the cocoon, as the larvae survive without healthy development and the productivity of the cocoons grown. This results in a 20.0% reduction in its average weight and a 23.3% decrease in the weight of the shell. At the same time, on farms where mulberry leaves are in short supply, the cocoon yield per box of worms is 15-30 kilograms, and the quality is 14% lower. This is due to the fact that the growth of mulberry silkworms as a result of the inability of the silk gland to accumulate enough silk fluid due to underdevelopment and the satiety of the mulberry leaf significantly affect the activity of the silk gland. Feeding silkworms with high-nutrient varieties of mulberry leaves increased silkworm size by 6.9-10.1%, viability by 2.5%, total cocoons, yield by 4.5%, yield by 8 kg. In particular, a sharp change in the air temperature of the worm relative to the norm (25-26 °C) weakens the activity of the silk gland due to disruption of the growth and development process of worms. Due to the reduction of the amount of silk liquid synthesized, the viability of worms is 6.5%, the weight of the cocoon is 61.5-64.4%, the weight of the silk shell is 73.7-74.5%, the yield is 8-10 kg, and the variety is 6-12%. decrease has been scientifically proven.

Fitm2 is required for ER homeostasis and normal function of murine liver

The endoplasmic reticulum (ER)-resident protein fat storage-inducing transmembrane protein 2 (FIT2) catalyzes acyl-CoA cleavage invitro and is required for ER homeostasis and normal lipid storage in cells. The gene encoding FIT2 is essential for the viability of mice and worms. Whether FIT2 acts as an acyl-CoA diphosphatase invivo and how this activity affects the liver, where the protein was discovered, are unknown. Here, we report that hepatocyte-specific Fitm2 knockout (FIT2-LKO) mice fed a chow diet exhibited elevated acyl-CoA levels, ER stress, and signs of liver injury. These mice also had more triglycerides in their livers than control littermates due, in part, to impaired secretion of triglyceride-rich lipoproteins and reduced capacity for fatty acid oxidation. We found that challenging FIT2-LKO mice with a high-fat diet worsened hepatic ER stress and liver injury but unexpectedly reversed the steatosis phenotype, similar to what is observed in FIT2-deficient cells loaded with fatty acids. Our findings support the model that FIT2 acts as an acyl-CoA diphosphatase invivo and is crucial for normal hepatocyte function and ER homeostasis in the murine liver.

Histopathological evaluation of Onchocerca volvulus nodules by microscopy and by digital image analysis for the study of macrofilaricidal drug efficacy.

Novel drugs or drug combinations that kill or permanently sterilize adult Onchocerca volvulus worms would be very helpful for treatment and elimination of onchocerciasis. In absence of a reliable biomarker for viable adult worms, histopathological assessment of worms within onchocercal nodules is a standard method to determine macrofilaricidal activity. The goal of the present study was to determine the agreement between two independent experts in the analysis of nodule sections and to assess the value of digital imaging as a means of standardizing the analysis. Two expert microscopists independently assessed 605 nodules by direct microscopy. At least two sections with two different stains hematoxylin & eosin (H&E, APR immunostain) of paraffin-embedded, ethanol-fixed whole-nodule cross-sections were analyzed. After variables were identified prone to observer discrepancies, we performed a second study to compare consolidated results for 100 nodules obtained by the two readers by microscopy and by analysis of scanned, high resolution digital images (20x magnification). The last data set analyzed was a quality panel of 100 nodules that has been previously examined by microscopy, and included additional immunostains for Wolbachia endobacteria. These slides were digitalized, read by the two assessors and results were compared with original microscopy results. The degree of agreement between assessors varied for different parameters. Agreement for female worm counts in nodules was approximately 80%, while agreement regarding female worm viability was 98%. There were no major differences observed between results obtained by microscopy or digital images. Good agreement for important parameters was also observed for the nodules of the quality panel. Nodule analysis by experienced microscopists was reproducible with regard to important parameters such as identification of living female worms or detection of normal embryogenesis. Assessments varied more for other parameters, and we recommend continued use of two independent readers for detailed analyzes. Analysis of scanned images provided similar results to direct microscopy. This facilitates training and comparison of nodule findings by readers in different locations. Analysis of high quality digital images that can be viewed remotely should improve the quality and availability of nodule assessments that are primary endpoints for onchocerciasis clinical trials.

Controlling htt protein aggregation to assess toxicity in Caenorhabditis elegans.

Aberrant aggregation of proteins into amyloid fibrils is implicated in a wide range of neurodegenerative disorders, including Alzheimer's, Parkinson's, and Huntington's diseases. Beyond fibrils, a heterogeneous mixture of intermediate aggregates, such as oligomers also form, and these oligomers are potent neurotoxic agents. Because of the transient nature of oligomers and that aggregation reactions result in a complex mixture of aggregate species, it has proven challenging to establish the toxic mechanism of action to specific aggregate species. This is particularly true when expressing aggregating proteins within cells or organisms. Using a mutant huntingtin protein (htt) that readily aggregates, a protocol was developed to introduce well-characterized aggregate species directly to C. elegans to assess toxicity. By precisely controlling aggregation conditions (i.e. temperature, time, and centrifugation), uniform populations of htt aggregates were obtained. These populations were characterized using atomic force microscopy and dynamic light scattering. These aggregates were fed to N2 C. elegans in a dose dependent manner using a 96 well microplate. The impact of this exposure on worm viability was measured using survival and thrashing assays. Both htt oligomers and fibrils were cytotoxic to worms in a dose dependent manner. Having established that these controlled aggregate populations were toxic, this approach can be applied to study how a variety of exogenous factors alters the impact of these aggregates on C. elegans.

Appearance of biological and productivity indicators of foreign breeds and hybrids of bombyx mori l. propagated in seed enterprises in different conditions

At the global level, special attention is paid to the care of the mulberry silkworm Bombyx mori L. in different climatic conditions, the adaptation of monovoltine breeds and hybrids to different ecological regions, and the production of high-quality raw silk that meets the requirements of the international standard 2A, 3A, 4A. being directed. Today, creating a new generation of mulberry silkworm breeds and hybrids and improving the technological properties of cocoons and silk raw materials obtained from them, determining the effects of various environmental factors and objectively evaluating the physical and mechanical properties of cocoons are considered as urgent problems on a global scale. Currently, some of the mulberry silkworm seeds, which are not enough for farms in our country, are imported from the People's Republic of China. The economic value of these imported elite and industrial hybrids, namely silkworm egg viability, worm viability, length of larval larval period, biological factors such as mulberry silkworm, cocoon and shell weight, cocoon yield, silkiness and cocoon productivity such as fertile and non-fertile cocoons. It is scientifically based on the extent to which the natural climatic conditions of the regions of Uzbekistan affect productivity indicators such as percentage, dry weight of cocoons, percentage of defective cocoons.

Characterization of glutamate carboxypeptidase 2 orthologs in trematodes

BackgroundGlutamate carboxypeptidase 2 (GCP2) belongs to the M28B metalloprotease subfamily encompassing a variety of zinc-dependent exopeptidases that can be found in many eukaryotes, including unicellular organisms. Limited information exists on the physiological functions of GCP2 orthologs in mammalian tissues outside of the brain and intestine, and such data are completely absent for non-mammalian species. Here, we investigate GCP2 orthologs found in trematodes, not only as putative instrumental molecules for defining their basal function(s) but also as drug targets.MethodsIdentified genes encoding M28B proteases Schistosoma mansoni and Fasciola hepatica genomes were analyzed and annotated. Homology modeling was used to create three-dimensional models of SmM28B and FhM28B proteins using published X-ray structures as the template. For S. mansoni, RT-qPCR was used to evaluate gene expression profiles, and, by RNAi, we exploited the possible impact of knockdown on the viability of worms. Enzymes from both parasite species were cloned for recombinant expression. Polyclonal antibodies raised against purified recombinant enzymes and RNA probes were used for localization studies in both parasite species.ResultsSingle genes encoding M28B metalloproteases were identified in the genomes of S. mansoni and F. hepatica. Homology models revealed the conserved three-dimensional fold as well as the organization of the di-zinc active site. Putative peptidase activities of purified recombinant proteins were assayed using peptidic libraries, yet no specific substrate was identified, pointing towards the likely stringent substrate specificity of the enzymes. The orthologs were found to be localized in reproductive, digestive, nervous, and sensory organs as well as parenchymal cells. Knockdown of gene expression by RNAi silencing revealed that the genes studied were non-essential for trematode survival under laboratory conditions, reflecting similar findings for GCP2 KO mice.ConclusionsOur study offers the first insight to our knowledge into M28B protease orthologs found in trematodes. Conservation of their three-dimensional structure, as well as tissue expression pattern, suggests that trematode GCP2 orthologs may have functions similar to their mammalian counterparts and can thus serve as valuable models for future studies aimed at clarifying the physiological role(s) of GCP2 and related subfamily proteases.Graphical

Chitin contributes to the formation of a feeding structure in a predatory nematode

Some nematode predators and parasites form teeth-like denticles that are histologically different from vertebrate teeth, but their biochemical composition remains elusive. Here, we show a role of chitin in the formation of teeth-like denticles in Pristionchus pacificus, a model system for studying predation and feeding structure plasticity. Pristionchus forms two alternative mouth morphs with one tooth or two teeth, respectively. The P.pacificus genome encodes two chitin synthases, with the highly conserved chs-2 gene being composed of 60 exons forming at least four isoforms. Generating CRISPR-Cas9-based gene knockouts, we found that Ppa-chs-2 mutations that eliminate the chitin-synthase domain are lethal. However, mutations in the C terminus result in viable but teethless worms, with severe malformation of the mouth. Similarly, treatment with the chitin-synthase inhibitor Nikkomycin Z also results in teethless animals. Teethless worms can feed on various bacterial food sources but are incapable of predation. High-resolution transcriptomics revealed that Ppa-chs-2 expression is controlled by the sulfatase-encoding developmental switch Ppa-eud-1. This study indicatesa key role of chitin in the formation of teeth-like denticles and the complex feeding apparatus in nematodes.

Direct Proteomic Detection and Prioritization of 19 Onchocerciasis Biomarker Candidates in Humans

Onchocerca volvulus, the causative agent of onchocerciasis, infects over 20 million people and can cause severe dermatitis and ocular conditions including blindness. Current treatments employed in mass drug administration programs do not kill adult female worms, and common diagnostic tests cannot reliably assess viability of adult worms. There is an urgent need for better diagnostic tests to facilitate monitoring the efficacy of new treatments and disease elimination efforts. Here, eight plasma samples collected from individuals infected with O. volvulus and seven from uninfected individuals were analyzed by MS/MS spectrometry to directly identify O. volvulus proteins present in infected but absent in uninfected control samples. This direct proteomic approach for biomarker discovery had not been previously employed for onchocerciasis. Among all detected proteins, 19 biomarker candidates were supported by two or more unique peptides, identified in the plasma of at least three O. volvulus-infected human samples and absent in all control samples. Comprehensive analysis and ranking of these candidates included detailed functional annotation and a review of RNA-seq gene expression profiles. Isotope-labeled standard peptides were run in parallel and validated MS/MS peptide identifications for 15 peptides from 11 of the 19 proteins, and two infected urine and one uninfected urine sample was used for additional validation. A major antigen/OVOC11613 was identified as the most promising candidate with eight unique peptides across five plasma samples and one urine sample. Additional strong candidates included OVOC1523/ATP synthase, OVOC247/laminin and OVOC11626/PLK5, and along with OVOC11613, and were also detected in urine samples from onchocerciasis patients. This study has identified a promising novel set of proteins that will be carried forward to develop assays that can be used for diagnosis of O. volvulus infections and for monitoring treatment efficacy.

Anti-Onchocercal Properties of Extracts of Scoparia dulcis and Cylicodiscus gabunensis.

The elimination of onchocerciasis is hampered by the absence of suitable drugs that are effective against adult filariae. This study is aimed at assessing the anti-onchocercal effects of extracts of Scoparia dulcis and Cylicodiscus gabunensis that could serve as drug leads against onchocerciasis. Different parts of the plants (Scoparia dulcis and Cylicodiscus gabunensis) were extracted with hexane, methylene chloride, and methanol. The extracts were tested in vitro against the bovine model parasite, Onchocerca ochengi. Adult female worm viability was determined biochemically by MTT/formazan colorimetry, while the adult male and microfilariae viability were determined by microscopy based on % inhibition of worm motility score. Cytotoxicity and acute toxicity of active extracts were tested on monkey kidney epithelial cells (LLC-MK2) and Balb/C mice, respectively. The hexane extract of Scoparia dulcis recorded the highest activity, with IC50s of 50.78 μg/ml on both adult male and female worms and 3.91 μg/ml on microfilariae. For Cylicodiscus gabunensis extract, the highest activity was seen with the methylene chloride extract, with IC50s of 50.78 μg/ml, 62.50 μg/ml, and 16.28 μg/ml on, respectively, adult male, female, and microfilariae. The 50% cytotoxic concentration on the LLC-MK2 cells was 31.25 μg/ml for the most active extracts. No acute toxicity was recorded for the extracts. Phytochemical analysis of the extracts revealed the presence of alkaloids, flavonoids, sterols, saponins, phenols, and glycosides. This study validates the traditional use of these plants in treating onchocerciasis and suggests S. dulcis and C. gabunensis as new potential sources for the isolation of anti-onchocerca lead compounds.

Evaluation of the mechanism of action of albendazole on adult rat lungworm (Angiostrongyluscantonensis)

Albendazole is considered the anthelmintic of choice for the management of rat lungworm disease (neuroangiostrongyliasis), due to its broad spectrum of nematocidal activity and its ability to cross the blood-brain barrier. Albendazole binds to β-tubulins, preventing their polymerization into microtubules, thereby corrupting the cascade of cell division at metaphase, which ultimately leads to the death of individual cells and eventually the death of the parasite. Inhibition of microtubule formation will also hinder the axoplasmic transport system, affecting the neuronal activities of the parasite. While this mechanism has been explicated in other parasitic and non-parasitic nematodes, it has never been evaluated in Angiostrongylus cantonensis. This study evaluates the antimitotic effects of albendazole sulphoxide (active metabolite) on the microtubules of adult A. cantonensis using the tubulin polymerization assay and measures its effects on worm viability using the colorimetric MTT assay. Three different concentrations of albendazole (62.5 μM, 250 μΜ, and 1 mM) were evaluated. We saw a statistically significant dose-dependent reduction in the band intensity of polymerized tubulins (or microtubules) (P = 0.019), suggesting that albendazole imparts its antimitotic effect in a dose-dependent manner. Similarly, our MTT assay showed a dose-dependent decrease in formazan intensity (proportional to cell viability), suggesting that the rate of nematocidal activity of albendazole is also proportional to its concentration. In compiling the results from both these experiments, a correlation between the microtubule assembly and worm viability is evident.

Evaluation of Non-Invasive Thermal Imaging for Detection of Viability of Onchocerciasis Worms.

Onchocerciasis is causing blindness in over half a million people in the world today. Drug development for the disease is crippled as there is no way of measuring effectiveness of the drug without an invasive procedure. Drug efficacy measurement through assessment of viability of onchocerca worms requires the patients to undergo nodulectomy which is invasive, expensive, time-consuming, skill-dependent, infrastructure dependent and lengthy process. In this paper, we discuss the first-ever study that proposes use of machine learning over thermal imaging to non-invasively and accurately predict the viability of worms. The key contributions of the paper are (i) a unique thermal imaging protocol along with pre-processing steps such as alignment, registration and segmentation to extract interpretable features (ii) extraction of relevant semantic features (iii) development of accurate classifiers for detecting the existence of viable worms in a nodule. When tested on a prospective test data of 30 participants with 48 palpable nodules, we achieved an Area Under the Curve (AUC) of 0.85. Clinical Relevance- This is the first ever research effort of using thermal imaging in the assessment of viability of onchocerca worms and it resulted in a very high specificity>95% which makes it a promising modality to pursue further.