Abstract:
Setaria digitata resides in the abdominal cavity of ungulates and are generally
nonpathogenic in their natural host, cattle. However, transmission of infective larvae into
non-permissive hosts such as sheep, goats or horses can result in cerebrospinal
nematodiasis (CNS). CNS is a neuropathological disorder that results in major dysfunctions
of the central nervous system, producing symptoms of motor weakness, ataxia, lumbar
paralysis and ultimate death of infected animals. Therefore, this disease poses a serious
threat to livestock farming in tropical countries. Further, Setaria digitata can also infect
humans and cause abscesses, allergic reactions, enlarged lymph nodes, eye lesions and
lung inflammation showing gradual adaptation to humans. siRNA mediated RNA
interference (RNAi) is considered to be a promising reverse-genetic tool to study gene
functions. In this study, we used siRNA technique to specifically silence the expression of
S. digitata novel parasitic nematode-specific gene (SDNG). SDNG has been proven to be
specific for animal parasitic nematodes. It was found to be abundantly expressed in
longitudinal muscles, reproductive systems and during embryogenesis, in all stages of the
life cycle of S. digitata. siRNA was generated using the DNA fragments resulting from PCR
amplification of plasmid vector containing SDNG. This was carried out using forward and
reverse primer combinations (four primer pairs; SD1F/1R, SD2F/2R, SD3F/3R, SD4F/4R)
containing T7 promoter sequences. dsRNA was synthesized using T7 RNA polymerase.
They were cleaved to 21-23mer siRNA fragments using shortcut RNAse III, and labeled
with Cy3 labeling reagents. siRNA so generated was used to treat adult worms (40μg/ml
siRNA, 4hrs per day for 4 days) and microfilariae (10μg/ml siRNA, 3hrs per day for one
day) in RPMI culture medium containing 10% FBS, 30 μg/ml Streptomycin, 2.5 μg/ml
Amphotericin B without FCS in a CO2 incubator in the presence of 5% CO2 and at 37 ºC.
The visualization of siRNA treated adult worms and microfilariae under the fluorescent
microscope revealed uptake and localization of Cy3-labeled siRNA by S. digitata indicating
that dsRNA is taken up by S. digitata which can be used for siRNA mediated RNA
interference to study the functional role of S. digitata genes. This is the first demonstration
of siRNA uptake by S. digitata.
