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Abstract
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
Highlights
The cryopreservation of filarial nematodes has been studied for nearly 70 years
The three objectives of this study were: (i) to establish a suitable method for D. immitis mf cryopreservation by recovering viable mf after thawing, collect Third-stage larvae (L3s) from mosquitoes that had been fed the thawed mf; (ii) to confirm the infectivity of the L3s obtained from mosquitoes in ferrets and dogs; and (iii) to determine the reproductive fitness of adult heartworms that developed from the L3s following reestablishment in dogs
Adults derived from Freshly collected microfilariae (FCmf) were recovered at a higher rate and were generally larger, based on worm length and weight, than adults resulting from Cryopreserved microfilariae (CPmf)
Summary
The cryopreservation of filarial nematodes has been studied for nearly 70 years 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. 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. The intent is to reduce the pain, suffering and distress on animals that may result from diseases induced to study disease progression, and the use of scientific procedures needed to develop novel treatments and preventatives for heartworm disease
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