低温保存犬恶丝虫微丝蚴，在蚊子和脊椎动物宿主中复苏并完成生命周期。

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

机构信息

Zoetis, Veterinary Medicine Research and Development, 333 Portage St, Kalamazoo, MI, 49007, USA.

出版信息

Parasit Vectors. 2021 Jul 16;14(1):367. doi: 10.1186/s13071-021-04839-7.

DOI:10.1186/s13071-021-04839-7

PMID:34271978

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC8283926/

Abstract

BACKGROUND

The 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.

METHODS

A 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.

RESULTS

Fewer 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.

CONCLUSIONS

The 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.

摘要

背景

对丝虫线虫的冷冻保存研究已经进行了近 70 年。这些研究主要通过在有能力的昆虫载体中接种后微丝蚴（mf）的存活和发育到第三期幼虫（L3）来检查冷冻保存方法的效果。只有一项研究报告称，从冷冻保存的库存中完全重建了一种丝虫线虫（Brugia malayi）在有能力的脊椎动物宿主中的生命周期。在此之前的研究基础上，开发了一种冷冻保存方法来冷冻保存犬心丝虫，Dirofilaria immitis 的 mf。

方法

分别使用 6%和 4mM 的二甲基亚砜（DMSO）和聚乙烯吡咯烷酮（PVP）作为冷冻保护剂的组合，可获得 mf 的可接受的解冻后存活，这些 mf 在埃及伊蚊中发育成 L3。从冷冻保存和新鲜收集的 mf 中发育而来的 L3s 接种到雪貂和犬中，并在足够的接种后时间评估其发育为成年心丝虫的情况。

结果

与从新鲜收集的 mf 中获得的 L3s 相比，从冷冻保存的 mf 中获得的成年心丝虫数量较少，并且前者的重量和长度较小。从冷冻保存的 mf 来源的 L3s 感染的犬中，循环 mf 的出现时间和密度相似，与从新鲜收集的 mf 中感染的犬相似。从冷冻保存的 mf 中获得的成虫已经存活并在犬中产生了 3 年以上的有活力的 mf。在接种后 2 年和 3.5 年，从犬中回收的 L3s 中有大约 60%发育成成虫。