Filarial Nematode Brugia Research Articles

Cryogenic preservation of Dirofilaria immitis microfilariae, reactivation and completion of the life-cycle in the mosquito and vertebrate hosts

BackgroundThe cryopreservation of filarial nematodes has been studied for nearly 70 years. Largely, these studies examined the effectiveness of cryopreservation methods by using the post-thaw survival of microfilariae (mf) and the development to third-stage larvae (L3s) following inoculation into a competent insect vector. Only one study reported complete reestablishment of a filarial nematode (Brugia malayi) life-cycle in a competent vertebrate host from cryopreserved stock. Expanding on this previous research, a cryopreservation method was developed to cryopreserve the mf of the dog heartworm, Dirofilaria immitis.MethodsA combination of cryoprotectants, dimethyl sulfoxide (DMSO) and polyvinyl pyrolidone (PVP) at 6% and 4 mM, respectively, provided acceptable post-thaw survival of mf that developed into L3s in Aedes aegypti. L3s developed from cryopreserved and freshly collected mf in mosquitoes were inoculated into ferrets and dogs and were assessed after a sufficient duration post-inoculation for development into adult heartworms.ResultsFewer adult heartworms derived from cryopreserved stocks of mf were recovered from ferrets compared to adult heartworms derived from freshly collected mf, and the former were smaller by weight and length. The onset of patency (circulating mf) occurred at similar post-inoculation time points and at similar mf densities in dogs infected with L3s sourced from cryopreserved stocks or freshly collected mf. Adults derived from cryopreserved mf have survived and produced viable mf for more than 3 years in dogs. Approximately 60% of inoculated L3s were recovered as adults from dogs at 2 and 3.5 years post-inoculation.ConclusionsThe results from these direct comparisons demonstrate that cryopreserved mf can develop into L3s in vector mosquitoes and that these L3s are infective to both dogs and ferrets, where they undergo normal development into adult worms. These worms are able to mate and produce viable mf and complete the heartworm lifecycle in dog.Graphical

Thioredoxin from Brugia malayi: defining a 16-kilodalton class of thioredoxins from nematodes.

Thioredoxins are a family of small redox proteins that undergo NADPH-dependent reduction by thioredoxin reductase. This results in a supply of reducing equivalents that cells use in a wide variety of biological reactions, which include maintaining reduced forms of the enzymes important for protection against damage from high-energy oxygen radicals, the regulation of transcription factor activity, and the inhibition of apoptosis. Here we report on a new member of the thioredoxin family of proteins from the filarial nematode Brugia malayi, Bm-TRX-1, which defines a new subclass of 16-kDa thioredoxins that occur widely in nematodes, including Caenorhabditis elegans. In addition to being larger than the thioredoxins found in mammalian and bacterial species, the putative active site sequence of Bm-TRX-1, WCPPC, does not conform to the highly conserved WCGPC reported for thioredoxins from mammals to bacteria. Interestingly, an allelic form of Bm-TRX-1 was identified with an active site sequence WCPQC, which appears to be unique to the thioredoxins from filarial species. Bm-TRX-1 was between 98% and 35% identical to thioredoxins from other nematodes and approximately 20% identical to the thioredoxins from mammals and Escherichia coli. Bm-TRX-1 was constitutively transcribed throughout the B. malayi life cycle, and Bm-TRX protein was detectable in somatic extracts and excretory-secretory products from adults and microfilariae. Recombinant Bm-TRX-1 had thiodisulfide reductase activity, as measured by the reduction of insulin, and protected DNA from the nicking activity of oxygen radicals. Overexpression of Bm-TRX-1 in a human monocyte cell line negatively regulated tumor necrosis factor alpha-induced p38 mitogen-activated protein kinase activity, suggesting a possible role of the 16-kDa Bm-TRX-1 in immunomodulation.