{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,11,6]],"date-time":"2026-11-06T23:45:01Z","timestamp":1794008701394,"version":"4.8.0"},"reference-count":32,"publisher":"Springer Science and Business Media LLC","issue":"6","license":[{"start":{"date-parts":[[2025,6,1]],"date-time":"2025-06-01T00:00:00Z","timestamp":1748736000000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"},{"start":{"date-parts":[[2025,6,3]],"date-time":"2025-06-03T00:00:00Z","timestamp":1748908800000},"content-version":"vor","delay-in-days":2,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"}],"content-domain":{"domain":["link.springer.com"],"crossmark-restriction":false},"short-container-title":["Parasitol Res"],"published-print":{"date-parts":[[2025,6]]},"abstract":"<jats:title>Abstract<\/jats:title>\n          <jats:p>\n            <jats:italic>Dirofilaria immitis<\/jats:italic>, also known as canine heartworm, contains an endosymbiont, <jats:italic>Wolbachia<\/jats:italic>, in all life stages. The antibiotic, doxycycline, has been incorporated into heartworm treatment protocols to eliminate <jats:italic>Wolbachia<\/jats:italic>. Previous studies indicate that subsequent infection cannot be established using viable third-stage larvae (L3) developed from doxycycline-treated microfilariae (mf). The stages in which the development of larvae is impacted by doxycycline remain unknown. We examined the impact of doxycycline on the third-stage to fourth-stage larval molt, as it is the first molt of <jats:italic>D. immitis<\/jats:italic> after it invades the vertebrate host. Microfilaremic blood was collected weekly from <jats:italic>D. immitis\u2013<\/jats:italic>infected dogs with or without doxycycline treatment at 10\u00a0mg\/kg as recommended by the American Heartworm Society. Blood was collected weekly until the end of doxycycline treatment. The blood was used for L3 production and mf isolation. <jats:italic>Wolbachia<\/jats:italic> levels in mf and L3 were measured using real-time quantitative PCR. L3 were cultured in vitro for 9\u00a0days to assess whether molting occurred. The Fisher\u2019s exact test and Bonferroni correction were used for statistical analysis. The molting of L3 from the doxycycline-treated groups did not show a significant difference compared to the L3 from the control group at weeks 0, 1, 2, 3, and 4. The <jats:italic>Wolbachia<\/jats:italic> levels in mf and L3 decreased starting from 7\u00a0days post-treatment and remained less than five percent of controls throughout the treatment. Doxycycline treatment can eliminate <jats:italic>Wolbachia<\/jats:italic> in both mf and subsequently developed L3. The molts of the mf to L3 in the mosquito and the L3 to L4 molt in vitro do not appear to be impacted by the reduction or elimination of <jats:italic>Wolbachia<\/jats:italic>.<\/jats:p>","DOI":"10.1007\/s00436-025-08506-z","type":"journal-article","created":{"date-parts":[[2025,6,3]],"date-time":"2025-06-03T08:46:19Z","timestamp":1748940379000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["In vitro molting of Dirofilaria immitis third-stage larvae derived from microfilariae collected from doxycycline-treated dogs"],"prefix":"10.1007","volume":"124","author":[{"given":"Yi","family":"Chu","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Elyssa","family":"Campbell","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michael","family":"Dzimianski","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Christopher C.","family":"Evans","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Cassan","family":"Pulaski","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kaori","family":"Sakamoto","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Andrew R.","family":"Moorhead","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2025,6,3]]},"reference":[{"key":"8506_CR1","first-page":"29","volume-title":"Dirofilariasis","author":"D Abraham","year":"1988","unstructured":"Abraham D (1988) Biology of Dirofilaria immitis. In: Atwell RB (ed) Dirofilariasis. CRC Press, pp 29\u201346"},{"issue":"1","key":"8506_CR2","doi-asserted-by":"publisher","first-page":"12","DOI":"10.1186\/1756-3305-5-12","volume":"5","author":"A Albers","year":"2012","unstructured":"Albers A, Esum ME, Tendongfor N, Enyong P, Klarmann U, Wanji S, Hoerauf A, Pfarr K (2012) Retarded Onchocerca volvulus L1 to L3 larval development in the Simulium damnosum vector after anti-wolbachial treatment of the human host. Parasit Vectors 5(1):12. https:\/\/doi.org\/10.1186\/1756-3305-5-12","journal-title":"Parasit Vectors"},{"key":"8506_CR3","unstructured":"American-Heartworm-Society. (2023) New American Heartworm Society Heartworm Incidence Map reveals upward trend in heartworm cases"},{"issue":"2","key":"8506_CR4","doi-asserted-by":"publisher","first-page":"357","DOI":"10.1016\/s0020-7519(98)00200-8","volume":"29","author":"C Bandi","year":"1999","unstructured":"Bandi C, McCall JW, Genchi C, Corona S, Venco L, Sacchi L (1999) Effects of tetracycline on the filarial worms Brugia pahangi and Dirofilaria immitis and their bacterial endosymbionts Wolbachia. Int J Parasitol 29(2):357\u2013364. https:\/\/doi.org\/10.1016\/s0020-7519(98)00200-8","journal-title":"Int J Parasitol"},{"issue":"6","key":"8506_CR5","doi-asserted-by":"publisher","first-page":"713","DOI":"10.1128\/aac.8.6.713","volume":"8","author":"M Barza","year":"1975","unstructured":"Barza M, Brown RB, Shanks C, Gamble C, Weinstein L (1975) Relation between lipophilicity and pharmacological behavior of minocycline, doxycycline, tetracycline, and oxytetracycline in dogs. Antimicrob Agents Chemother 8(6):713\u2013720. https:\/\/doi.org\/10.1128\/aac.8.6.713","journal-title":"Antimicrob Agents Chemother"},{"issue":"12","key":"8506_CR6","doi-asserted-by":"publisher","first-page":"1401","DOI":"10.1016\/j.ijpara.2008.03.002","volume":"38","author":"C Bazzocchi","year":"2008","unstructured":"Bazzocchi C, Mortarino M, Grandi G, Kramer LH, Genchi C, Bandi C, Genchi M, Sacchi L, McCall JW (2008) Combined ivermectin and doxycycline treatment has microfilaricidal and adulticidal activity against Dirofilaria immitis in experimentally infected dogs. Int J Parasitol 38(12):1401\u20131410. https:\/\/doi.org\/10.1016\/j.ijpara.2008.03.002","journal-title":"Int J Parasitol"},{"issue":"4","key":"8506_CR7","doi-asserted-by":"publisher","first-page":"160","DOI":"10.1053\/j.tcam.2011.07.001","volume":"26","author":"DD Bowman","year":"2011","unstructured":"Bowman DD, Mannella C (2011) Macrocyclic lactones and Dirofilaria immitis microfilariae. Top Companion Anim Med 26(4):160\u2013172. https:\/\/doi.org\/10.1053\/j.tcam.2011.07.001","journal-title":"Top Companion Anim Med"},{"issue":"12","key":"8506_CR8","doi-asserted-by":"publisher","first-page":"1457","DOI":"10.1016\/S0020-7519(02)00158-3","volume":"32","author":"M Casiraghi","year":"2002","unstructured":"Casiraghi M, McCall JW, Simoncini L, Kramer LH, Sacchi L, Genchi C, Werren JH, Bandi C (2002) Tetracycline treatment and sex-ratio distortion: a role for Wolbachia in the moulting of filarial nematodes? Int J Parasitol 32(12):1457\u20131468. https:\/\/doi.org\/10.1016\/S0020-7519(02)00158-3","journal-title":"Int J Parasitol"},{"issue":"12","key":"8506_CR9","doi-asserted-by":"publisher","first-page":"2467","DOI":"10.1101\/gr.138420.112","volume":"22","author":"AC Darby","year":"2012","unstructured":"Darby AC, Armstrong SD, Bah GS, Kaur G, Hughes MA, Kay SM, Koldkj\u00e6r P, Rainbow L, Radford AD, Blaxter ML, Tanya VN, Trees AJ, Cordaux R, Wastling JM, Makepeace BL (2012) Analysis of gene expression from the genome of a filarial nematode supports both metabolic and defensive roles within the symbiosis. Genome Res 22(12):2467\u20132477. https:\/\/doi.org\/10.1101\/gr.138420.112","journal-title":"Genome Res"},{"issue":"1","key":"8506_CR10","doi-asserted-by":"publisher","first-page":"39","DOI":"10.1186\/s13071-018-2631-0","volume":"11","author":"J Drake","year":"2018","unstructured":"Drake J, Wiseman S (2018) Increasing incidence of Dirofilaria immitis in dogs in USA with focus on the southeast region 2013\u20132016. Parasit Vectors 11(1):39. https:\/\/doi.org\/10.1186\/s13071-018-2631-0","journal-title":"Parasit Vectors"},{"key":"8506_CR11","doi-asserted-by":"publisher","unstructured":"Foster J, Ganatra M, Kamal I, Ware J, Makarova K, Ivanova N, Bhattacharyya A, Kapatral V, Kumar S, Posfai J, Vincze T, Ingram J, Moran L, Lapidus A, Omelchenko M, Kyrpides N, Ghedin E, Wang S, Goltsman E, . . . Slatko B (2005) The Wolbachia genome of Brugia malayi: endosymbiont evolution within a human pathogenic nematode. PLoS Biol 3(4):e121. https:\/\/doi.org\/10.1371\/journal.pbio.0030121","DOI":"10.1371\/journal.pbio.0030121"},{"key":"8506_CR12","unstructured":"FR3. (accessed 2024). Filariasis Research Reagent Resource Center Protocols. Retrieved October, 25 from https:\/\/sites.uwosh.edu\/filariasis-center\/protocols\/parasite-protocols\/"},{"key":"8506_CR13","doi-asserted-by":"crossref","unstructured":"Kotani T, Powers KG (1982) Developmental stages of Dirofilaria immitis in the dog. Am J Vet Res 43(12):2199\u20132206. https:\/\/www.ncbi.nlm.nih.gov\/pubmed\/7165165","DOI":"10.2460\/ajvr.1982.43.12.2199"},{"issue":"3","key":"8506_CR14","doi-asserted-by":"publisher","first-page":"191","DOI":"10.1016\/j.vetpar.2008.09.014","volume":"158","author":"L Kramer","year":"2008","unstructured":"Kramer L, Grandi G, Leoni M, Passeri B, McCall J, Genchi C, Mortarino M, Bazzocchi C (2008) Wolbachia and its influence on the pathology and immunology of Dirofilaria immitis infection. Vet Parasitol 158(3):191\u2013195. https:\/\/doi.org\/10.1016\/j.vetpar.2008.09.014","journal-title":"Vet Parasitol"},{"key":"8506_CR15","doi-asserted-by":"publisher","first-page":"113617","DOI":"10.1016\/j.foodres.2023.113617","volume":"174","author":"DY Lee","year":"2023","unstructured":"Lee DY, Yun SH, Lee SY, Lee J Jr, Mariano E, Joo ST, Choi I, Choi JS, Kim GD, Lee J, Choi S-H, Hur SJ (2023) Analysis of commercial fetal bovine serum (FBS) and its substitutes in the development of cultured meat. Food Res Int 174:113617. https:\/\/doi.org\/10.1016\/j.foodres.2023.113617","journal-title":"Food Res Int"},{"issue":"4","key":"8506_CR16","doi-asserted-by":"publisher","first-page":"402","DOI":"10.1006\/meth.2001.1262","volume":"25","author":"KJ Livak","year":"2001","unstructured":"Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25(4):402\u2013408. https:\/\/doi.org\/10.1006\/meth.2001.1262","journal-title":"Methods"},{"key":"8506_CR17","doi-asserted-by":"publisher","first-page":"1208301","DOI":"10.3389\/fmicb.2023.1208301","volume":"14","author":"AE Marriott","year":"2023","unstructured":"Marriott AE, Dagley JL, Hegde S, Steven A, Fricks C, DiCosty U, Mansour A, Campbell EJ, Wilson CM, Gusovsky F, Ward SA, Hong WD, O\u2019Neill P, Moorhead A, McCall S, McCall JW, Taylor MJ, Turner JD (2023) Dirofilariasis mouse models for heartworm preclinical research. Front Microbiol 14:1208301. https:\/\/doi.org\/10.3389\/fmicb.2023.1208301","journal-title":"Front Microbiol"},{"issue":"4","key":"8506_CR18","doi-asserted-by":"publisher","first-page":"361","DOI":"10.1016\/j.vetpar.2011.01.022","volume":"176","author":"JW McCall","year":"2011","unstructured":"McCall JW, Kramer L, Genchi C, Guerrero J, Dzimianski MT, Supakorndej P, Mansour A, McCall SD, Supakorndej N, Grandi G, Carson B (2011) Effects of doxycycline on early infections of Dirofilaria immitis in dogs. Vet Parasitol 176(4):361\u2013367. https:\/\/doi.org\/10.1016\/j.vetpar.2011.01.022","journal-title":"Vet Parasitol"},{"issue":"1\u20132","key":"8506_CR19","doi-asserted-by":"publisher","first-page":"5","DOI":"10.1016\/j.vetpar.2014.09.023","volume":"206","author":"JW McCall","year":"2014","unstructured":"McCall JW, Kramer L, Genchi C, Guerrero J, Dzimianski MT, Mansour A, McCall SD, Carson B (2014) Effects of doxycycline on heartworm embryogenesis, transmission, circulating microfilaria, and adult worms in microfilaremic dogs. Vet Parasitol 206(1\u20132):5\u201313. https:\/\/doi.org\/10.1016\/j.vetpar.2014.09.023","journal-title":"Vet Parasitol"},{"issue":"1","key":"8506_CR20","doi-asserted-by":"publisher","first-page":"199","DOI":"10.1186\/s13071-023-05704-5","volume":"16","author":"JW McCall","year":"2023","unstructured":"McCall JW, DiCosty U, Mansour A, Fricks C, McCall S, Dzimianski MT, Carson B (2023) Inability of Dirofilaria immitis infective larvae from mosquitoes fed on blood from microfilaremic dogs during low-dose and short-treatment regimens of doxycycline and ivermectin to complete normal development in heartworm na\u00efve dogs. Parasit Vectors 16(1):199. https:\/\/doi.org\/10.1186\/s13071-023-05704-5","journal-title":"Parasit Vectors"},{"key":"8506_CR21","doi-asserted-by":"publisher","unstructured":"McCall JW, Genchi C, Kramer LH, Guerrero J, Venco L (2008) Chapter 4 Heartworm disease in animals and humans. In Advances in parasitology (Vol. 66, pp. 193\u2013285). Academic Press. https:\/\/doi.org\/10.1016\/S0065-308X(08)00204-2","DOI":"10.1016\/S0065-308X(08)00204-2"},{"key":"8506_CR22","unstructured":"Mostafa Z (Accessed 2024) Zamanian Lab Docx - Lab Protocols. Retrieved October 25 from https:\/\/www.zamanianlab.org\/ZamanianLabDocs\/labmanual\/"},{"issue":"Suppl 2","key":"8506_CR23","doi-asserted-by":"publisher","first-page":"515","DOI":"10.1186\/s13071-017-2446-4","volume":"10","author":"CT Nelson","year":"2017","unstructured":"Nelson CT, Myrick ES, Nelson TA (2017) Clinical benefits of incorporating doxycycline into a canine heartworm treatment protocol. Parasit Vectors 10(Suppl 2):515. https:\/\/doi.org\/10.1186\/s13071-017-2446-4","journal-title":"Parasit Vectors"},{"key":"8506_CR24","unstructured":"Nelson CT (2023) Chapter 111. Heartworm and related nematodes. In Sykes JE (Ed.), Greene's Infectious Diseases of the Dog and Cat. Elsevier, Inc. https:\/\/www.sciencedirect.com\/science\/article\/pii\/B9780323509343001117. (Reprinted from Fifth Edition)"},{"issue":"15","key":"8506_CR25","doi-asserted-by":"publisher","first-page":"e2120003119","DOI":"10.1073\/pnas.2120003119","volume":"119","author":"S Quek","year":"2022","unstructured":"Quek S, Cook DAN, Wu Y, Marriott AE, Steven A, Johnston KL, Ford L, Archer J, Hemingway J, Ward SA, Wagstaff SC, Turner JD, Taylor MJ (2022) Wolbachia depletion blocks transmission of lymphatic filariasis by preventing chitinase-dependent parasite exsheathment. Proc Natl Acad Sci U S A 119(15):e2120003119. https:\/\/doi.org\/10.1073\/pnas.2120003119","journal-title":"Proc Natl Acad Sci U S A"},{"issue":"3","key":"8506_CR26","doi-asserted-by":"publisher","first-page":"605","DOI":"10.1645\/0022-3395(2002)088[0605:IVEOAO]2.0.CO;2","volume":"88","author":"R Rao","year":"2002","unstructured":"Rao R, Weil GJ (2002) In vitro effects of antibiotics on Brugia malayi worm survival and reproduction. J Parasitology 88(3):605\u2013611, 607. https:\/\/doi.org\/10.1645\/0022-3395(2002)088[0605:IVEOAO]2.0.CO;2","journal-title":"J Parasitology"},{"issue":"1","key":"8506_CR27","doi-asserted-by":"publisher","first-page":"77","DOI":"10.1016\/S0014-4894(02)00019-X","volume":"101","author":"RU Rao","year":"2002","unstructured":"Rao RU, Moussa H, Weil GJ (2002) Brugia malayi: effects of antibacterial agents on larval viability and development in vitro. Experimental Parasitology 101(1):77\u201381. https:\/\/doi.org\/10.1016\/S0014-4894(02)00019-X","journal-title":"Experimental Parasitology"},{"issue":"3","key":"8506_CR28","doi-asserted-by":"publisher","first-page":"587","DOI":"10.1016\/j.ijpddr.2018.09.002","volume":"8","author":"BJ Reaves","year":"2018","unstructured":"Reaves BJ, Wallis C, McCoy CJ, Lorenz WW, Rada B, Wolstenholme AJ (2018) Recognition and killing of Brugia malayi microfilariae by human immune cells is dependent on the parasite sample and is not altered by ivermectin treatment. Int J Parasitol Drugs Drug Resist 8(3):587\u2013595. https:\/\/doi.org\/10.1016\/j.ijpddr.2018.09.002","journal-title":"Int J Parasitol Drugs Drug Resist"},{"issue":"1","key":"8506_CR29","doi-asserted-by":"publisher","first-page":"671","DOI":"10.1186\/s13071-018-3264-z","volume":"11","author":"MD Savadelis","year":"2018","unstructured":"Savadelis MD, Day KM, Bradner JL, Wolstenholme AJ, Dzimianski MT, Moorhead AR (2018) Efficacy and side effects of doxycycline versus minocycline in the three-dose melarsomine canine adulticidal heartworm treatment protocol. Parasit Vectors 11(1):671. https:\/\/doi.org\/10.1186\/s13071-018-3264-z","journal-title":"Parasit Vectors"},{"issue":"4","key":"8506_CR30","doi-asserted-by":"publisher","first-page":"265","DOI":"10.1006\/expr.2000.4525","volume":"95","author":"HL Smith","year":"2000","unstructured":"Smith HL, Rajan TV (2000) Tetracycline inhibits development of the infective-stage larvae of filarial nematodes in vitro. Experimental Parasitology 95(4):265\u2013270. https:\/\/doi.org\/10.1006\/expr.2000.4525","journal-title":"Experimental Parasitology"},{"issue":"4","key":"8506_CR31","doi-asserted-by":"publisher","first-page":"520","DOI":"10.1111\/cmi.12084","volume":"15","author":"MJ Taylor","year":"2013","unstructured":"Taylor MJ, Voronin D, Johnston KL, Ford L (2013) Wolbachia filarial interactions. Cell Microbiol 15(4):520\u2013526. https:\/\/doi.org\/10.1111\/cmi.12084","journal-title":"Cell Microbiol"},{"issue":"6","key":"8506_CR32","doi-asserted-by":"publisher","first-page":"920","DOI":"10.1021\/tx700002f","volume":"20","author":"BA Wagner","year":"2007","unstructured":"Wagner BA, Teesch LM, Buettner GR, Britigan BE, Burns CP, Reszka KJ (2007) Inactivation of anthracyclines by serum heme proteins. Chem Res Toxicol 20(6):920\u2013926. https:\/\/doi.org\/10.1021\/tx700002f","journal-title":"Chem Res Toxicol"}],"container-title":["Parasitology Research"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/link.springer.com\/content\/pdf\/10.1007\/s00436-025-08506-z.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/link.springer.com\/article\/10.1007\/s00436-025-08506-z\/fulltext.html","content-type":"text\/html","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/link.springer.com\/content\/pdf\/10.1007\/s00436-025-08506-z.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,7,3]],"date-time":"2025-07-03T08:41:37Z","timestamp":1751532097000},"score":1,"resource":{"primary":{"URL":"https:\/\/link.springer.com\/10.1007\/s00436-025-08506-z"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,6]]},"references-count":32,"journal-issue":{"issue":"6","published-print":{"date-parts":[[2025,6]]}},"alternative-id":["8506"],"URL":"https:\/\/doi.org\/10.1007\/s00436-025-08506-z","relation":{},"ISSN":["0932-0113","1432-1955"],"issn-type":[{"value":"0932-0113","type":"print"},{"value":"1432-1955","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,6]]},"assertion":[{"value":"5 March 2025","order":1,"name":"received","label":"Received","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"21 May 2025","order":2,"name":"accepted","label":"Accepted","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"3 June 2025","order":3,"name":"first_online","label":"First Online","group":{"name":"ArticleHistory","label":"Article History"}},{"order":1,"name":"Ethics","group":{"name":"EthicsHeading","label":"Declarations"}},{"value":"All procedures performed in this study were approved by the University of Georgia Institutional Animal Care and Use Committee guidelines using approved Animal Use Protocol A2022 04\u2013006.","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":"The authors declare no competing interests.","order":4,"name":"Ethics","group":{"name":"EthicsHeading","label":"Competing interests"}}],"article-number":"59"}}