Evidence-based investigations into the ethnoveterinary use of Mimosa pudica L. (Fabaceae) as an anthlemintic

Purpose: To investigate the toxicity, clinical outcome and anthelmintic effects of M. pudica in vitro and in vivo.Methods: Dried leaves of M. pudica were extracted using 70 % methanol cold maceration method. Acute toxicity inquiry was evaluated using Lorke’s method. Anthelmintic effects were investigated in vitro using the egg hatch assay and in vivo using Heligmosomoides bakeri experimentally infected adult albino mice. Coprological and haematological parameters were recorded during the experiment while the serological analysis and post mortem worm burden were assessed at the conclusion of the research.Results: No mortality was recorded in oral acute toxicity test up to a dose of 5000 mg/kg. A probit-log analysis of the percentage egg hatch of the extract and albendazole gave lethal concentration 50 (LC50) values of 1.160 and -1.042, respectively. A reduction in worm count was observed in all the extracttreated groups post mortem, with the maximum extract group having the least worm count (p < 0.05). Treatment with extract resulted in improvement in the haematological parameters. Serum chemistry revealed no significant differences (p > 0.05) in alanine aminotransferase and blood urea nitrogen in all groups. However, a dose-dependent increases in the total protein and albumin was observed.Conclusion: These results show that although M. pudica has weak anthelmintic effects compared to albendazole (standard anthelmintic), in vivo and in vitro, at the doses used in this study, nonetheless, it reduces worm burden and improves haematologic parameters, serum total protein, albumin and overall weight gain of the treated mice. Thus, increased doses may be effective in anthelmintic chemotherapy. Keywords: Mimosa pudica, Anthelmintic, Toxicity, Heligmosomoides bakeri, Ethnoveterinary medicine, Anthelmintic

The development of spicules in Heligmosomoides bakeri (Nematoda, Heligmosomidae)

The spicules of male parasitic nematodes are key morphological features, which vary between species in shape and length and are used often for species identification. However, little is known about spicules and particularly if/how their length varies during growth. We first assessed the degree of variation in spicule length of male Heligmosomoides bakeri 21 days post infection (PI), and then in two follow-up experiments measured spicule lengths at half daily/daily intervals between days 6 and 14 PI. Mean spicule length in 21-day worms was 0.518 mm with a range of 94 μm, and variation between the two spicules of individual worms from 2 to 32 μm. Spicules were first detectable on day 6–6.5, after which their lengths increased until day 7 PI (mean = 0.61 and 0.59). This was followed by significant contraction, initially relatively quickly over the following 48 h and then more slowly over a longer period, stabilizing by days 10–14, with only minor further reduction in length. We conclude that the length of spicules varies significantly over the first few days after they have formed, and, consequently, the age of worms is an important factor for consideration when spicule lengths are measured for experimental/diagnostic or taxonomical purposes.

The effect of conventional preservatives on spicule length of Heligmosomoides bakeri (Nematoda, Heligmosomidae)

Nematode spicules vary in shape and size even between closely related species and, therefore, constitute key characters in nematode taxonomy for distinguishing between species. Spicules are seldom measured on fresh specimens, but rather at some time after extraction from culled hosts and after a period of preservation of the worms in chemical fixatives or by freezing. We carried out two experiments to assess the effects of freezing in Hanks’ balanced salt solution, 70% or 80% ethanol and 10% formalin (both of the latter at room temperature and after storage at −80°C) on spicule length of Heligmosomoides bakeri at two time intervals after extraction from mice (Experiment 1, one and four weeks; Experiment 2, one and four months). In Experiment 1, no significant differences were detected, although there was some variation between treatments and over time. In Experiment 2, spicule length varied significantly between treatments and over time, the greatest shrinkage being in 80% ethanol and the least in 10% formalin. However, overall variation in spicule length was very limited, accounting for no more than 5.03% change in length over time and 4.95% between treatments at any of the periods of assessment. Therefore, while whole nematodes can shrivel and shrink in preservatives, making many measurements unreliable, our data indicated that spicule lengths are very little changed by preservation techniques over time, and so spicule length remains as a reliable taxonomic character.

Disentangling sRNA-Seq data to study RNA communication between species

Many organisms exchange small RNAs (sRNAs) during their interactions, that can target or bolster defense strategies in host–pathogen systems. Current sRNA-Seq technology can determine the sRNAs present in any symbiotic system, but there are very few bioinformatic tools available to interpret the results. We show that one of the biggest challenges comes from sequences that map equally well to the genomes of both interacting organisms. This arises due to the small size of the sRNAs compared to large genomes, and because a large portion of sequenced sRNAs come from genomic regions that encode highly conserved miRNAs, rRNAs or tRNAs. Here, we present strategies to disentangle sRNA-Seq data from samples of communicating organisms, developed using diverse plant and animal species that are known to receive or exchange RNA with their symbionts. We show that sequence assembly, both de novo and genome-guided, can be used for these sRNA-Seq data, greatly reducing the ambiguity of mapping reads. Even confidently mapped sequences can be misleading, so we further demonstrate the use of differential expression strategies to determine true parasite-derived sRNAs within host cells. We validate our methods on new experiments designed to probe the nature of the extracellular vesicle sRNAs from the parasitic nematode Heligmosomoides bakeri that get into mouse intestinal epithelial cells.

Ovicidal and Larvicidal Activities of Saba senegalensis (A.DC) Pichon (Apocynaceae) Extracts and Fractions on Heligmosomoides bakeri (Nematoda, Heligmosomatidae)

Aims: To investigate ovicidal and larvicidal activities of an aqueous decoction (AD) and hydroethanolic macerate (HEM) extracts and fractions of the leaves of Saba senegalensis. Study Design: In vitro, the ovicidal and larvicidal activities of AD and HEM extracts and fractions of the leaves of Saba senegalensis on the eggs and larvae (L1) of Heligmosomoides bakeri. Place and Duration of Study: The experiment was conducted at the department of Medicine and Traditional Pharmacopeia-Pharmacy (MEPHATRA-PH) of Institute of Research in Health Sciences (IRSS) between June 2015 and December 2016. Methodology: The phytochemical groups of the extract and fractions of Saba senegalensis were determined by a colorimetric and Thin Layer Chromatography methods. The eggs were obtained from feces of mice deliberately infected and the larvae from the eggs were incubated at 25 ±2℃ for 72 hours. Eggs and larvae were exposed to increasing concentrations (100; 625; 1250; 2500; 3750 µg/mL) of the different extracts, 48 hours and 24 hours for the eggs and larvae respectively. Distilled water and DMSO 0.1% were used as negative controls while albendazole and levamisole were used as positive controls. Results: The phytochemical groups of interest are the tannins, saponins, flavonoids and triterpenes. The negative control had given 2.16% of egg hatch inhibition and 0% of larvae mortality mean while the positive control had given 100% in both cases. The extracts inhibited eggs hatching and affected larval survival. Pharmacological effects were concentration-dependent. The ovicidal and larvicidal activity of HEM is more interesting than that of AD with an Emax = 95.60% and an IC50 = 390 µg/mL. It is the same for the larvicidal activity with Emax = 100% and an LC50 = 900 µg/mL. However, the differences were not statistically significant. Conclusion: These results show the ovicidal and larvicidal properties of the S. senegalensis leaves.

The Effects of Bonny Light Crude Oil (Blco) on the Helminth Parasite Heligmosomoides Bakeri (Polygrus) of Mice.

This study examined the effect of crude oil on Heligmosomoides bakeri in albino mice. A total of 35 albino mice of 5-8 weeks were used for this study. The mice were randomly divided into five groups (A, B, C, D and E) of 5 mice each and infected with 0.13ml of H. bakeri larvae. Mice in the first two groups (A and B) were given crude oil per os at the concentrations of 0.2 and 0.05 mg/ml respectively. Those in group C were given Albendazole and group D infected but not treated. Group E was used as a naïve control group. Two other groups F and G were used for toxicity test. All mice were observed for clinical signs and symptoms of abnormality all through the study and all parameters were assessed following standard procedures for 5 weeks. Administration of crude oil per os to mice in groups A and B at the dose levels used in this study showed no significant (p>0.05) changes in PCV, body weight and fecal egg counts of the mice. There was a significant (p<0.05) difference in larval mortality test between the crude oil treated groups compared to the control group by their movement (if normal, moving or dead i.e. no observed motion within 10 mins). The Albendazole group C had the least worm burden and fecal egg count compared to the groups treated with different concentration of Crude oil. In conclusion, it may be suggested that heavily infected animals may not respond to treatment with crude oil against nematodes which discredit the unorthodox folklore medicine for parasitic treatment by some rural. Its effect on the worms appeared to be transient.

Secretion of an Argonaute protein by a parasitic nematode and the evolution of its siRNA guides

Extracellular RNA has been proposed to mediate communication between cells and organisms however relatively little is understood regarding how specific sequences are selected for export. Here we describe a specific Argonaute protein (exWAGO) that is secreted in extracellular vesicles (EVs) released by the gastrointestinal nematode Heligmosomoides bakeri, at multiple copies per EV. Phylogenetic and gene expression analyses demonstrate exWAGO orthologues are highly conserved and abundantly expressed in related parasites but highly diverged in free-living genus Caenorhabditis. We show that the most abundant small RNAs released from the nematode parasite are not microRNAs as previously thought, but rather secondary small interfering RNAs (siRNAs) that are produced by RNA-dependent RNA Polymerases. The siRNAs that are released in EVs have distinct evolutionary properties compared to those resident in free-living or parasitic nematodes. Immunoprecipitation of exWAGO demonstrates that it specifically associates with siRNAs from transposons and newly evolved repetitive elements that are packaged in EVs and released into the host environment. Together this work demonstrates molecular and evolutionary selectivity in the small RNA sequences that are released in EVs into the host environment and identifies a novel Argonaute protein as the mediator of this.