A histochemical study of the Nras/let-60 activity in filarial nematodes

BACKGROUND: Control and elimination of filarial pathogens is a central focus of major global health efforts directed at parasitic diseases of developing countries. Accomplishment of these goals would be markedly enhanced by the enhanced destruction of the adult stage of filariae. The identification of new, more quantitative biomarkers that correlate with mortality or chemotherapeutic damage to adult filariae, would greatly facilitate, for example, the development of new macrofilaricides. METHODS: An immunocytochemical approach using an antibody against human Nras was used to identify and detect changes in the nematode homolog let-60 that is associated with cell growth and maintenance. Single Onchocerca volvulus nodules were removed from each of 13 patients treated with ivermectin (as part of a community-wide mass drug administration programme), and from each of 13 untreated individuals; these 26 nodules were stained with the anti-Nras antibody. The localization and degree of positivity of Nras/let-60 staining were assessed subjectively and compared between the two groups; the positivity of staining was also quantified, using image analysis, in a subgroup of these nodules. In addition, the specific morphological association between Nras/let-60 and the Wolbachia endosymbiont present in these parasites was also observed in 4 additional filarial species using an anti-Wolbachia surface protein (WSP) antibody under light and confocal microscopy. RESULTS: Nras/let-60 is present in many structures within the adult female worms. A statistically significant decrease in the general staining intensity of Nras/let-60 was observed in adult female O. volvulus treated with ivermectin when compared with parasites from untreated patients. Nras/let-60 staining was frequently observed to be co-localized with WSP in O.volvulus, Brugia malayi, Litomosoides sigmodontis and Dirofilaria immitis. Nras/let60 is also present in Onchocerca ochengi. CONCLUSION: Nras/let-60, as detected by immunocytochemical staining, is decreased in ivermectin-treated adult female O. volvulus relative to untreated control specimens, suggesting a suppressive effect of ivermectin on the overall biochemical activity of these parasites. Co-localization of Nras/let-60 and WSP suggests the possibility that the endosymbiont utilizes this nematode protein as part of a mutualistic relationship. Nras/let60 appears to be a useful biomarker for assessing the health of filariae. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13071-015-0947-6) contains supplementary material, which is available to authorized users

The Effect of In Vitro Cultivation on the Transcriptome of Adult Brugia malayi

Infections with filarial worms cause serious physical impairment and affect tens of millions of people in tropical and subtropical countries. To better understand the biology and phar- macology of these parasites, Brugia malayi is often used as a model. This parasite can be maintained in the laboratory in Mongolian jirds, enabling researchers to test drugs in vivoand in vitro, among other studies. The effects of removing worms from their hosts and cul- turing them may affect many aspects of their physiology, including response to drugs, but the extent to which the worms undergo changes during culture has remained unknown. Using deep RNA sequencing and bioinformatics tools, we examined the global transcriptomic profile of B. malayi females at four different time points over 5 days in culture. Focusing on genes that are differentially expressed at various time points, we observed a general perturbation of the expression profile between dissection from the host and receipt after shipment. The expression of several genes remained changed at the end of the experi- ment, after 5 days under controlled conditions; in particular, genes encoding cuticle colla- gens were prominently represented and strongly overexpressed

The Effects of Ivermectin on <i>Brugia malayi</i> Females <i>In Vitro</i>: A Transcriptomic Approach

<div><p>Background</p><p>Lymphatic filariasis and onchocerciasis are disabling and disfiguring neglected tropical diseases of major importance in developing countries. Ivermectin is the drug of choice for mass drug administration programs for the control of onchocerciasis and lymphatic filariasis in areas where the diseases are co-endemic. Although ivermectin paralyzes somatic and pharyngeal muscles in many nematodes, these actions are poorly characterized in adult filariae. We hypothesize that paralysis of pharyngeal pumping by ivermectin in filariae could result in deprivation of essential nutrients, especially iron, inducing a wide range of responses evidenced by altered gene expression, changes in metabolic pathways, and altered developmental states in embryos. Previous studies have shown that ivermectin treatment significantly reduces microfilariae release from females within four days of exposure <i>in vivo</i>, while not markedly affecting adult worms. However, the mechanisms responsible for reduced production of microfilariae are poorly understood.</p><p>Methodology/Principal Findings</p><p>We analyzed transcriptomic profiles from <i>Brugia malayi</i> adult females, an important model for other filariae, using RNAseq technology after exposure in culture to ivermectin at various concentrations (100 nM, 300 nM and 1 μM) and time points (24, 48, 72 h, and 5 days). Our analysis revealed drug-related changes in expression of genes involved in meiosis, as well as oxidative phosphorylation, which were significantly down-regulated as early as 24 h post-exposure. RNA interference phenotypes of the orthologs of these down-regulated genes in <i>C</i>. <i>elegans</i> include “maternal sterile”, “embryonic lethal”, “larval arrest”, “larval lethal” and “sick”.</p><p>Conclusion/Significance</p><p>These changes provide insight into the mechanisms involved in ivermectin-induced reduction in microfilaria output and impaired fertility, embryogenesis, and larval development.</p></div

Gene Ontology enrichment analysis of top 15 biological processes at 100 nM IVM exposure for 48 h.

<p>Gene Ontology enrichment analysis of top 15 biological processes at 100 nM IVM exposure for 48 h.</p

Tissue damage score following recovery of FLBZ-exposed <i>B</i>. <i>malayi</i> females from naïve jirds.

<p>A, B. Worms incubated for 24 hr prior to transplantation and recovered from jirds for 5 days, 4 weeks or 8 weeks after. Data is a combination of two experiments. In experiment one worms transplanted into each of three jirds for all treatment groups were maintained for 5 days or 4 weeks. Worms transplanted into each of three jirds per treatment group in experiment two were maintained for 4 weeks or 8 weeks. The 4 week data were combined for presentation purposes. A minimum of 25 worms were assessed histologically C, D. Worms incubated for 6, 12, or 24 hr prior to recovery at 8 weeks. Worms were transplanted into each of three jirds per treatment group, following the appropriate duration of <i>in vitro</i> exposure to FLBZ. The number of worms assessed histologically ranged from 11 to 39. Damage scored as minor (1), moderate (2), severe (3), or no damage (0) by two methods: A, C. Individual section scoring method; B, D. Classical histopathological survey method.</p

Differentially expressed genes in each time point comparison, with the highest fold-changes.

<p>A log₂ fold-change of +3.5 and -3.5 was applied, respectively. GO terms were retrieved from Nematode.net. Green indicates down-regulation and red, up-regulation.</p

Correlation between RNAseq and qPCR data from 5 genes at different time points.

<p>Fold-change values of the selected genes are displayed in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004311#pntd.0004311.s003" target="_blank">S1 Table</a>. The correlation coefficient between RNAseq (x-axis) and qPCR (y-axis) data (log₂ fold-change) analyzed by the Pearson test was 0.9961 with a statistical significance <i>p</i><0.01.</p

Study design.

<p>At each time point, two or three groups of 10 worms were washed, flash-frozen (represented by lightning bolt) and used for RNA extraction. IVM: ivermectin.</p

Picard alignment summary metrics.

<p>Summary of the sequencing and mapping of the data to the <i>B</i>. <i>malayi</i> transcriptome.</p

Venn diagram showing the number of overlapping genes across pairwise comparisons of each time point compared to baseline.

<p>Venn diagram was created using Venny 2.0.</p