{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,22]],"date-time":"2026-10-22T22:26:58Z","timestamp":1792708018175,"version":"4.5.0"},"reference-count":24,"publisher":"Springer Science and Business Media LLC","issue":"1","license":[{"start":{"date-parts":[[2021,7,16]],"date-time":"2021-07-16T00:00:00Z","timestamp":1626393600000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"},{"start":{"date-parts":[[2021,7,16]],"date-time":"2021-07-16T00:00:00Z","timestamp":1626393600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"}],"content-domain":{"domain":["link.springer.com"],"crossmark-restriction":false},"short-container-title":["Parasites Vectors"],"published-print":{"date-parts":[[2021,12]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:sec>\n                <jats:title>Background<\/jats:title>\n                <jats:p>The cryopreservation of filarial nematodes has been studied for nearly 70\u00a0years. 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 (<jats:italic>Brugia malayi<\/jats:italic>) 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, <jats:italic>Dirofilaria immitis<\/jats:italic>.<\/jats:p>\n              <\/jats:sec><jats:sec>\n                <jats:title>Methods<\/jats:title>\n                <jats:p>A combination of cryoprotectants, dimethyl sulfoxide (DMSO) and polyvinyl pyrolidone (PVP) at 6% and 4\u00a0mM, respectively, provided acceptable post-thaw survival of mf that developed into L3s in <jats:italic>Aedes aegypti<\/jats:italic>. 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.<\/jats:p>\n              <\/jats:sec><jats:sec>\n                <jats:title>Results<\/jats:title>\n                <jats:p>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\u00a0years in dogs. Approximately 60% of inoculated L3s were recovered as adults from dogs at 2 and 3.5\u00a0years post-inoculation.<\/jats:p>\n              <\/jats:sec><jats:sec>\n                <jats:title>Conclusions<\/jats:title>\n                <jats:p>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.<\/jats:p>\n              <\/jats:sec><jats:sec>\n                <jats:title>Graphical Abstract<\/jats:title>\n                \n              <\/jats:sec>","DOI":"10.1186\/s13071-021-04839-7","type":"journal-article","created":{"date-parts":[[2021,7,16]],"date-time":"2021-07-16T09:03:07Z","timestamp":1626426187000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":10,"title":["Cryogenic preservation of Dirofilaria immitis microfilariae, reactivation and completion of the life-cycle in the mosquito and vertebrate hosts"],"prefix":"10.1186","volume":"14","author":[{"given":"Erich W.","family":"Zinser","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tom L.","family":"McTier","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nicole S.","family":"Kernell","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Debra J.","family":"Woods","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2021,7,16]]},"reference":[{"key":"4839_CR1","doi-asserted-by":"publisher","first-page":"193","DOI":"10.1016\/S0065-308X(08)00204-2","volume":"66","author":"JW McCall","year":"2008","unstructured":"McCall JW, Genchi C, Kramer LH, Guerrero J, Venco L. Heartworm disease in animals and humans. Adv Parasitol. 2008;66:193\u2013285.","journal-title":"Adv Parasitol"},{"issue":"4","key":"4839_CR2","doi-asserted-by":"publisher","first-page":"186","DOI":"10.1053\/j.tcam.2011.09.004","volume":"26","author":"TG Geary","year":"2011","unstructured":"Geary TG, Bourguinat C, Prichard RK. Evidence for macrocyclic lactone anthelmintic resistance in Dirofilaria immitis. Top Companion Anim Med. 2011;26(4):186\u201392.","journal-title":"Top Companion Anim Med"},{"key":"4839_CR3","doi-asserted-by":"publisher","first-page":"191","DOI":"10.1186\/s13071-016-1476-7","volume":"9","author":"BL Blagburn","year":"2016","unstructured":"Blagburn BL, Arther RG, Dillon AR, Butler JM, Bowles JV, von Simson C, et al. Efficacy of four commercially available heartworm preventive products against the JYD-34 laboratory strain of Dirofilaria immitis. Parasites Vectors. 2016;9:191.","journal-title":"Parasites Vectors"},{"issue":"1","key":"4839_CR4","doi-asserted-by":"publisher","first-page":"339","DOI":"10.1186\/s13071-020-04178-z","volume":"13","author":"K Kryda","year":"2020","unstructured":"Kryda K, Holzmer S, Everett WR, McCall JW, Mahabir SP, McTier TL, et al. Preventive efficacy of four or six monthly oral doses of 24 microg\/kg moxidectin compared to six monthly doses of Heartgard(R) Plus or Interceptor(R) Plus against macrocyclic lactone-resistant heartworm (Dirofilaria immitis) strains in dogs. Parasites Vectors. 2020;13(1):339.","journal-title":"Parasites Vectors"},{"issue":"3\u20134","key":"4839_CR5","doi-asserted-by":"publisher","first-page":"167","DOI":"10.1016\/j.vetpar.2015.04.002","volume":"210","author":"C Bourguinat","year":"2015","unstructured":"Bourguinat C, Lee AC, Lizundia R, Blagburn BL, Liotta JL, Kraus MS, et al. Macrocyclic lactone resistance in Dirofilaria immitis: Failure of heartworm preventives and investigation of genetic markers for resistance. Vet Parasitol. 2015;210(3\u20134):167\u201378.","journal-title":"Vet Parasitol"},{"key":"4839_CR6","doi-asserted-by":"publisher","first-page":"494","DOI":"10.1186\/s13071-014-0494-6","volume":"7","author":"CN Pulaski","year":"2014","unstructured":"Pulaski CN, Malone JB, Bourguinat C, Prichard R, Geary T, Ward D, et al. Establishment of macrocyclic lactone resistant Dirofilaria immitis isolates in experimentally infected laboratory dogs. Parasites Vectors. 2014;7:494.","journal-title":"Parasites Vectors"},{"issue":"Suppl 2","key":"4839_CR7","doi-asserted-by":"publisher","first-page":"482","DOI":"10.1186\/s13071-017-2429-5","volume":"10","author":"TL McTier","year":"2017","unstructured":"McTier TL, Six RH, Pullins A, Chapin S, McCall JW, Rugg D, et al. Efficacy of oral moxidectin against susceptible and resistant isolates of Dirofilaria immitis in dogs. Parasites Vectors. 2017;10(Suppl 2):482.","journal-title":"Parasites Vectors"},{"key":"4839_CR8","doi-asserted-by":"publisher","first-page":"27","DOI":"10.1017\/S0022149X00020307","volume":"34","author":"AE Taylor","year":"1960","unstructured":"Taylor AE. The development of Dirofilaria immitis in the mosquito Aedes aegypti. J Helminthol. 1960;34:27\u201338.","journal-title":"J Helminthol"},{"issue":"1","key":"4839_CR9","doi-asserted-by":"publisher","first-page":"138","DOI":"10.4269\/ajtmh.1983.32.138","volume":"32","author":"RC Lowrie Jr","year":"1983","unstructured":"Lowrie RC Jr. Cryopreservation of the microfilariae of Brugia malayi, Dirofilaria corynodes, and Wuchereria bancrofti. 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All three studies were conducted according to state and national regulations regarding animal welfare.","order":2,"name":"Ethics","group":{"name":"EthicsHeading","label":"Ethics approval and consent to participate"}},{"value":"Not applicable.","order":3,"name":"Ethics","group":{"name":"EthicsHeading","label":"Consent for publication"}},{"value":"EWZ, TLM, and DJW are current employees of Zoetis LLC. NSK is a current employee of Priority One, a contractor for Zoetis.","order":4,"name":"Ethics","group":{"name":"EthicsHeading","label":"Competing interests"}}],"article-number":"367"}}