Abstract
BackgroundmicroRNAs (miRNAs), a class of short, non-coding RNA can be found in a highly stable, cell-free form in mammalian body fluids. Specific miRNAs are secreted by parasitic nematodes in exosomes and have been detected in the serum of murine and dog hosts infected with the filarial nematodes Litomosoides sigmodontis and Dirofilaria immitis, respectively. Here we identify extracellular, parasite-derived small RNAs associated with Onchocerca species infecting cattle and humans.MethodsSmall RNA libraries were prepared from total RNA extracted from the nodule fluid of cattle infected with Onchocerca ochengi as well as serum and plasma from humans infected with Onchocerca volvulus in Cameroon and Ghana. Parasite-derived miRNAs were identified based on the criteria that sequences unambiguously map to hairpin structures in Onchocerca genomes, do not align to the human genome and are not present in European control serum.ResultsA total of 62 mature miRNAs from 52 distinct pre-miRNA candidates were identified in nodule fluid from cattle infected with O. ochengi of which 59 are identical in the genome of the human parasite O. volvulus. Six of the extracellular miRNAs were also identified in sequencing analyses of serum and plasma from humans infected with O. volvulus. Based on sequencing analysis the abundance levels of the parasite miRNAs in serum or plasma range from 5 to 127 reads/per million total host miRNA reads identified, comparable to our previous analyses of Schistosoma mansoni and L. sigmodontis miRNAs in serum. All six of the O. volvulus miRNAs identified have orthologs in other filarial nematodes and four were identified in the serum of mice infected with L. sigmodontis.ConclusionsWe have identified parasite-derived miRNAs associated with onchocerciasis in cattle and humans. Our results confirm the conserved nature of RNA secretion by diverse nematodes. Additional species-specific small RNAs from O. volvulus may be present in serum based on the novel miRNA sequences identified in the nodule fluid. In our analyses comparison to European control serum illuminates the scope for false-positives, warranting caution in criteria that should be applied to identification of biomarkers of infection.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-015-0656-1) contains supplementary material, which is available to authorized users.
Highlights
MicroRNAs, a class of short, non-coding RNA can be found in a highly stable, cell-free form in mammalian body fluids
O. ochengi small RNAs are present in bovine nodule fluid RNA from the fluid of nodules of cattle infected with O. ochengi was analysed for small RNA content using the Agilent Bioanalyzer
Six O. volvulus miRNAs were identified in human plasma, all of which are identical to those found in O. ochengi nodule fluid, and four of which are identical to those found in serum of mice infected with the related filarial nematode L. sigmodontis (Figure 3)
Summary
MicroRNAs (miRNAs), a class of short, non-coding RNA can be found in a highly stable, cell-free form in mammalian body fluids. Specific miRNAs are secreted by parasitic nematodes in exosomes and have been detected in the serum of murine and dog hosts infected with the filarial nematodes Litomosoides sigmodontis and Dirofilaria immitis, respectively. Parasite-derived small RNAs associated with Onchocerca species infecting cattle and humans. We showed that miRNAs are packaged within vesicles secreted by the gastrointestinal nematode Heligmosomoides polygyrus and that these derive from the intestine of the nematode. These secreted vesicles (and their cargoes) suppress Th2 innate immune responses in vivo and the miRNAs within them are transferred to host cells in vitro [9]. We characterize the extracellular, parasite-derived miRNAs associated with the important human disease onchocerciasis
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