We previously reported fatal infection of a captive Bornean orangutan with metacestodes of a novel taeniid tapeworm, Versteria sp. New data implicate mustelids as definitive hosts of these tapeworms in North America. At least 2 parasite genetic lineages circulate in North America, representing separate introductions from Eurasia.Peer reviewe

Clyde, Victoria L.

Gendron-Fitzpatrick, Annette

Goldberg, Tony L.

Hoberg, Eric P.

Lauck, Michael

Lavikainen, Antti

Lee, Laura M.

Nakao, Minoru

O'Connor, David H.

Sibley, Samuel D.

Stuchin, Margot

Wallace, Roberta S.

Emerging Infectious Diseases

PubMed

Laura M. Lee

Roberta S. Wallace

Victoria L. Clyde

Annette Gendron-Fitzpatrick

Samuel D. Sibley

Margot Stuchin

Michael Lauck

David H. O’Connor

Minoru Nakao

Antti Lavikainen

Eric P. Hoberg

Tony L. Goldberg

Crossref

Definitive Hosts of<i>Versteria</i>Tapeworms (Cestoda: Taeniidae) Causing Fatal Infection in North America

Helsingin yliopiston digitaalinen arkisto

Laura M. Lee, Roberta S. Wallace,  Victoria L. Clyde, Annette Gendron-Fitzpatrick, Samuel D. Sibley, Margot Stuchin,  Michael Lauck, David H. O’Connor,  Minoru Nakao, Antti Lavikainen,  Eric P. Hoberg, Tony L. GoldbergWe	previously	reported	fatal	infection	of	a	captive	Bornean	orangutan	with	metacestodes	of	a	novel	taeniid	tapeworm,	Versteria	 sp.	 New	 data	 implicate	 mustelids	 as	 definitive	hosts	of	these	tapeworms	in	North	America.	At	least	2	para-site	genetic	lineages	circulate	in	North	America,	represent-ing	separate	introductions	from	Eurasia.	Taeniid tapeworms (Cestoda: Taeniidae) comprise 4 proposed genera: Taenia, Echinococcus, Hydatigera, and Versteria (1). Until recently, genetic data were absent for Versteria sp. in North America. However, in 2014, we reported an unusual case of fatal metacestode (larval stage of tapeworm) infection in a captive Bornean orangutan (Pongo pygmaeus); the causative agent was identified as a novel Versteria genotype (2).As previously described (2), the orangutan was born at a zoo in Colorado, USA, and was rejected by his birth mother. Approximately 10 months later, he was transported to the Milwaukee County Zoo in Milwaukee, Wisconsin, USA, for adoption by a surrogate mother. At ≈5 years of age, he died unexpectedly from acute respiratory distress due to disseminated infection with an unknown agent. A combination of metagenomics and gene-specific DNA se-quencing revealed the etiologic agent to be a previously unknown Versteria lineage in its larval form.We obtained wild mustelids (carnivores of the fam-ily Mustelidae) from Colorado, near where the animal was born, and Wisconsin, near where the animal died. We targeted mustelids because they are definitive hosts of V. mustelae tapeworms in Europe (3) and V. brachyacantha tapeworms in Africa (4). Colorado samples were submis-sions to the Denver Museum of Nature and Science, and Wisconsin samples were obtained from a local fur trapper. We also examined mustelids from Oregon, USA, as part of an ongoing investigation of Versteria spp. tapeworms in the Nearctic region and Eurasia.From 4 mustelids from Colorado (1 otter [Lontra ca-nadensis], 2 ermine [Mustela erminea], and 1 mink [Neovi-son vison]), we recovered 1 adult tapeworm from a female ermine collected 178 km from where the orangutan was born. From 17 mustelids from Wisconsin (1 mink, 5 long-tailed weasels [M. frenata], and 11 ermine), we recovered 1 adult tapeworm from a male ermine collected 56 km from where the orangutan died. From 17 mustelids from Oregon (1 mink, 1 ermine, and 15 long-tailed weasels), we recov-ered 1 adult tapeworm from an adult mink of unknown sex. All tapeworm specimens were fragmented, lacking intact scolices, or both, which prevented complete morphologic description; however, microscope examination of mature segments from the Wisconsin and Oregon specimens re-vealed structures consistent with those of parasites in the genus Versteria (Figure 1).We sequenced 396 bp of the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene from the 3 new adult tape-worm specimens according to previously published meth-ods (2). The sequences of the tapeworms from the Colo-rado ermine and the Oregon mink were 99.5% and 99.2% similar, respectively, to the sequence from the orangutan, placing these new specimens confidently within the same Versteria lineage (Figure 2). By contrast, the sequence from the tapeworm from the Wisconsin ermine was only 90.7% similar to the sequence from the orangutan, mak-ing it a heretofore unrecognized lineage that clusters more closely with parasites from Eurasia (Figure 2).To investigate the hosts from which adult tapeworms were recovered, we amplified and sequenced 751 bp of the mustelid cytochrome b (cytb) gene, using DNA ex-tracts from adult tapeworm material. To do this, we used previously published methods (7,8) with modified PCR primers MVZ45_must_F (5′-CAGTNATAGCAACAG-CATTCATAGG-3′) and MVZ14_must_R (5′-GCTCTC-CATTTTTGGTTTACAAGAC-3′). This effort was suc-cessful, demonstrating that adult tapeworm material Definitive Hosts of Versteria Tapeworms  (Cestoda: Taeniidae) Causing Fatal Infection  in North America	 Emerging	Infectious	Diseases	•	www.cdc.gov/eid	•	Vol.	22,	No.	4,	April	2016	 707Author	affiliations:	University	of	Wisconsin–Madison,	Madison,	Wisconsin,	USA	(L.M.	Lee,	A.	Gendron-Fitzpatrick,	S.D.	Sibley,	 M.	Lauck,	D.H.	O’Connor);	Milwaukee	County	Zoo,	Milwaukee,	Wisconsin,	USA	(R.S.	Wallace,	V.L.	Clyde,	A.	Gendron-Fitzpatrick);	 Colorado	State	University,	Fort	Collins,	Colorado,	USA	 (M.	Stuchin);	Wisconsin	National	Primate	Research	Center,	Madi-son	(M.	Lauck,	D.H.	O’Connor,	T.L.	Goldberg);	Asahikawa	Medical	University,	Asahikawa,	Hokkaido,	Japan	(M.	Nakao);	University	of	Helsinki,	Helsinki,	Finland	(A.	Lavikainen);	United	States	National	Parasite	Collection,	Beltsville,	Maryland,	USA	(E.P.	Hoberg)DOI:	http://dx.doi.org/10.3201/eid2204.151446DISPATCHEScontains sufficient host-associated DNA for such analy-ses. The resulting sequences indicated that the ermine from Colorado and Wisconsin (GenBank accession nos. KT223030 and KT223032, respectively) belonged to the continental clade, which is broadly distributed across the Nearctic region (9). The sequence for the Oregon mink (GenBank accession no. KT223031) closely matched pub-lished sequences for other mink from North America but was otherwise phylogeographically uninformative.We next assessed whether parasites of the genus Ver-steria were present in wild carnivores on and near the grounds of the Milwaukee County Zoo. We conducted systematic searches of the zoo grounds (i.e., outside of en-closures) for carnivore feces. We also trapped carnivores, using live traps baited with fish, and then immediately released them, collecting any feces deposited in the trap, and we collected feces from mink from a local wildlife rehabilitation center. These efforts yielded 51 samples (3 from skunks [Mephitis mephitis], 3 from coyotes [Canis latrans], 6 from long-tailed weasels, 8 from mink, and 31 from raccoons [Procyon lotor]), which we examined by microscope, using standard flotation and sedimentation methods to identify parasite eggs (Table 1). We then used the ZR Fecal DNA MiniPrep kit (Zymo Research, Irvine, CA, USA) to extract DNA directly from feces samples and amplified and sequenced the cox1 gene (2). Resulting sequences were unambiguous and revealed a diversity of parasites (not only taeniids, and in some cases representing putative novel lineages), but none matched Versteria spp. (Table 2).Our results demonstrate that the genus Versteria is a species complex in North America. The new lineage iden-tified in Wisconsin clusters with parasites formerly known only from Eurasia (Figure 2). Preliminary assessments suggest that this same lineage infects ermine as far away as the Northwest Territories (NWT) of Canada (E.P. Ho-berg et al., unpub. data) and that the lineage responsible for fatal infection of an orangutan (2) also infects musk-rats (Ondatra zibethicus) in Idaho, USA, and the NWT (A. Lavikainen et al., unpub. data). To the extent that current sampling reflects the distribution of Versteria lineages in North America, there appears to be a western lineage (rep-resented by Colorado, Oregon, and Idaho, and the NWT) and a northern continental lineage (represented by Wiscon-sin and the NWT), with sympatry in the NWT. We note that this pattern parallels the phylogeography of ermine in North America, perhaps reflecting postglacial expansion of host and parasite after the Pleistocene (9). More Versteria lineages will likely be found, as evidenced by a novel lin-eage in zokor (Eospalax baileyi, a fossorial rodent) recently described from Tibet (10) (Figure 2). Versteria lineages have clearly entered the Nearctic region from the Palearctic region at least twice, probably reflecting Beringian biogeo-graphic processes of faunal expansion that are important drivers of the evolution of mammals and the organisms parasitizing them (11).Our findings shed light on the origins of the infection that proved fatal to the orangutan. We found an adult Ver-steria sp. tapeworm with a nearly identical DNA sequence in Colorado, where the orangutan was born; however, a Versteria sp. tapeworm from Wisconsin, where the orang-utan died, was genetically divergent. Moreover, we found no evidence of Versteria tapeworms on the grounds of the Milwaukee County Zoo or nearby. Taeniid metacestodes encyst and can remain dormant for years before asexual multiplication (12). We therefore suspect that the orang-utan became infected where it was born (Colorado) and 708	 Emerging	Infectious	Diseases	•	www.cdc.gov/eid	•	Vol.	22,	No.	4,	April	2016Figure 1.	Microscope	image	of	a	mature	segment	of	an	adult	Versteria	sp.	tapeworm	recovered	from	an	ermine	in	Wisconsin,	USA	(original	magnification	×10).	Characteristic	reproductive	structures	are	visible,	including	genital	pore	(G),	cirrus	sac	(C),	vagina	(V),	ovary	(O),	testes	(T),	uterine	stem	(U),	and	vitelline	gland	(VG).	Tapeworm	specimens	were	preserved	in	70%	ethanol	for	concurrent	morphologic	and	molecular	analyses.	A	series	of	proglottids	was	subsampled	from	each	worm	as	a	basis	for	sequencing;	remaining	strobila	was	stained,	cleared	and	mounted	in	Canada	balsam	as	permanent	vouchers	based	on	standard	methods	(5).	Specimens	are	deposited	in	the	Museum	of	Southwestern	Biology,	Parasitology	Division,	University	of	New	Mexico,	Albuquerque,	New	Mexico,	USA	(accession	no.	MSB	23169),	and	in	the	collections	of	the	Denver	Museum	of	Nature	and	Science,	Denver,	Colorado,	USA	(accession	no.	DZTM.3170).	Scale	bar	indicates	500	µm.	Hosts	of	Versteria Tapeworms	in	North	Americacarried the latent infection to where it died ≈4 years later (Wisconsin). This animal’s sudden progression to disease remains a mystery, perhaps indicating immune deficiency or another precipitating factor, consistent with reports of disseminated taeniid infection in other hosts (13).In general, our findings underscore that exotic animals in zoo settings are susceptible to infections harbored by lo-cal wildlife and that transport of such animals can compli-cate inferences about the origins of these locally acquired infections. We reiterate that taeniid tapeworms of the genus Versteria should be considered a threat to captive apes (2), and we recommend that wild mustelids, such as ermine and mink, be excluded or removed from the grounds of zoos where apes have access to outdoor environments. Given the close relationship between apes and humans, we also sug-gest increased vigilance for zoonotic infections.AcknowledgmentsWe are grateful to the staff and administration of the  Milwaukee County Zoo for their support, John Demboski 	 Emerging	Infectious	Diseases	•	www.cdc.gov/eid	•	Vol.	22,	No.	4,	April	2016	 709Figure 2.	Phylogenetic	tree	of	members	of	the	genus	Versteria	(Cestoda:	Taeniidae).	The	tree	was	constructed	from	a	DNA	sequence	alignment	of	cytochrome	c	oxidase	subunit	1	genes.	The	maximum-likelihood	method	was	used,	with	the	likeliest	model	of	molecular	evolution	(Hasegawa-Kishino-Yano	model	with	invariable	positions),	which	was	chosen	by	using	MEGA6	(6).	Numbers	next	to	branches	indicate	bootstrap	values	(%),	estimated	from	1,000	resamplings	of	the	data,	and	the	tree	is	rooted	at	the	midpoint	of	the	longest	branch.	Taxon	labels	indicate	parasite	species,	intermediate	or	definitive	host,	and	geographic	origin	(GenBank	accession	nos.	in	parentheses).	Bold	indicates	sequences	from	this	study	(from	adult	parasites	and	definitive	hosts).	Scale	bar	indicates	nucleotide	substitutions	per	site   Table 1.	Parasites	in	wild	carnivore	feces	samples	collected	on	the	grounds	of	and	near	the	Milwaukee	County	Zoo,	Milwaukee,	Wisconsin,	USA,	2014* Parasite Host,	%	positive Coyote,	n	=	3 Long-tailed	weasel,	n	=	6 Mink,	n	=	8 Raccoon,	n	=	31 Skunk,	n	=	3 Ascarid – – – 12.9 – Baylisascaris procyonis – – – 35.5 – Cestode 33.3 50.0 16.7 25.8 66.7 Coccidia 66.7 33.3 – 22.6 33.3 Cystoisospora spp. – – – 3.2 – Giardia spp. – – 16.7 25.8 – Hookworm – – – 12.9 33.3 Strongylid – 16.7 33.3 12.9 33.3 Trichuris spp. – – 33.3 22.6 – Other† 66.7 33.3 50.0 3.2 – *–,	not	present. †Other	parasites	were	unidentified	metastrongyles,	nematodes,	and	protozoans.  DISPATCHESand the Denver Museum of Nature and Science for access to mustelid specimens from Colorado, Katie Moriarty and Nicole Edmison for access to mustelid specimens from Oregon, Slade Noreen and Jorden Manasse for assistance with mustelid speci-mens from Wisconsin, and Daron Graves and Patricia Khan for  assistance with obtaining samples from the grounds of the Milwaukee County Zoo.This work was supported by the American Association of Zoological Veterinarians (award MSN173939). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Field collections in the Northwest Territories and broader explorations of diversity were conducted through the Collaborative Integrated Inventory of Biomes of the Arctic project, supported by the National Science Foundation (grants no. 0196095 and 0415668).Ms. Lee is a veterinary student who participated in this research as part of the University of Wisconsin-Madison School of Veterinary Medicine’s Summer Scholars Program. Her interests include wild-life medicine, small animal medicine, and infectious disease.References  1. Nakao M, Lavikainen A, Iwaki T, Haukisalmi V, Konyaev S, Oku Y, et al. Molecular phylogeny of the genus Taenia (Cestoda: Taeniidae): proposals for the resurrection of Hydatigera Lamarck, 1816 and the creation of a new genus Versteria. Int J Parasitol. 2013;43:427–37. http://dx.doi.org/10.1016/j.ijpara.2012.11.014  2. Goldberg TL, Gendron-Fitzpatrick A, Deering KM, Wallace RS, Clyde VL, Lauck M, et al. Fatal metacestode infection in Bornean orangutan caused by unknown Versteria species. Emerg Infect Dis. 2014;20:109–13. http://dx.doi.org/10.3201/eid2001.131191  3. Loos-Frank B. An up-date of Verster’s (1969) ‘Taxonomic revision of the genus Taenia Linnaeus’ (Cestoda) in table format. Syst Parasitol. 2000;45:155–84. http://dx.doi.org/10.1023/A:1006219625792  4. Baer JG, Fain A. Newly discovered Cestoda in Belgian Congo [in French]. Acta Trop. 1951;8:59–63.  5. Pritchard MH, Kruse GOW. The collection and preservation of  animal parasites. Lincoln (Nebraska): University of Nebraska Press; 1982.  6. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Mol Biol Evol. 2013;30:2725–9. http://dx.doi.org/10.1093/molbev/mst197  7. Smith MF, Patton JL. The diversification of South American murid rodents: evidence from mitochondrial DNA sequence data for the akodontine tribe. Biol J Linn Soc Lond. 1993;50:149–77.  http://dx.doi.org/10.1111/j.1095-8312.1993.tb00924.x  8. Fleming MA, Cook JA. Phylogeography of endemic ermine  (Mustela erminea) in southeast Alaska. Mol Ecol. 2002;11:795–807. http://dx.doi.org/10.1046/j.1365-294X.2002.01472.x  9. Dawson NG, Hope AG, Talbot SL, Cook JA. A multilocus  evaluation of ermine (Mustela erminea) across the Holarctic,  testing hypotheses of Pleistocene diversification in response to climate change. J Biogeogr. 2014;41:464–75. http://dx.doi.org/ 10.1111/jbi.1222110. Zhao F, Ma JY, Cai HX, Su JP, Hou ZB, Zhang TZ, et al. Molecular identification of Taenia mustelae cysts in subterranean rodent plateau zokors (Eospalax baileyi). Dongwuxue Yanjiu. 2014;35:313–8.11. Hoberg EP, Galbreath KE, Cook JA, Kutz SJ, Polley L.  Northern host–parasite assemblages: history and biogeography on the borderlands of episodic climate and environmental transition. Adv Parasitol. 2012;79:1–97. http://dx.doi.org/10.1016/ B978-0-12-398457-9.00001-912. Whitfield PJ, Evans NA. Parthenogenesis and asexual multiplica-tion among parasitic platyhelminths. Parasitology. 1983;86:121–60. http://dx.doi.org/10.1017/S003118200005087313. Hoberg EP, Ebinger W, Render JA. Fatal cysticercosis by Taenia crassiceps (Cyclophyllidea: Taeniidae) in a presumed  immunocompromised canine host. J Parasitol. 1999;85:1174–8. http://dx.doi.org/10.2307/3285685Address for correspondence: Tony L. Goldberg, University of Wisconsin-Madison, Department of Pathobiological Sciences, 1656 Linden Dr, Madison, WI 53706, USA; email: tony.goldberg@wisc.edu710	 Emerging	Infectious	Diseases	•	www.cdc.gov/eid	•	Vol.	22,	No.	4,	April	2016   Table 2. Parasites	identified	by	DNA	sequencing	of	the cox1 gene	in	samples	of	wild	carnivore	feces	collected	on	the	grounds	of	and	near	the	Milwaukee	County	Zoo, Milwaukee,	Wisconsin, USA,	2014* Host GenBank accession no.† Most	similar	to % Similarity‡ Mink KT223036 Alaria alata 91.2	(HM022221) Raccoon KT223037 Baylisascaris procyonis 100.0	(KC172104) Long-tailed	weasel KT223038 Parafilaroides normani 89.6	(KJ801815) Skunk KT223039 Taenia crassiceps 86.9	(EU544549) Mink KT223040 Toxascaris leonina 93.1	(JF780951) *Only	samples	with	parasite	eggs	resembling	those	of	cestodes	were	tested;	mink	samples	were	from	a	local	wildlife	rehabilitation	center.	Sequences	matching	noncestode	parasites	indicate	nonspecificity	of	PCR	primers;	results	do	not	exclude	the	possibility	of	mixed	infections.	cox1,	cytochrome	c	oxidase	subunit	1. †For parasite sequences newly generated during this study. ‡Nucleotide similarity to the most	similar	sequence	in	GenBank	(accession	no.	in	parentheses)	as	of	June	26,	2015.  

Definitive Hosts of Versteria Tapeworms (Cestoda : Taeniidae) Causing Fatal Infection in North America

We previously reported fatal infection of a captive Bornean orangutan with metacestodes of a novel taeniid tapeworm, Versteria sp. New data implicate mustelids as definitive hosts of these tapeworms in North America. At least 2 parasite genetic lineages circulate in North America, representing separate introductions from Eurasia

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Definitive Hosts of Versteria Tapeworms (Cestoda: Taeniidae) Causing Fatal Infection in North America

Definitive Hosts of Versteria Tapeworms (Cestoda : Taeniidae) Causing Fatal Infection in North America

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Helsingin yliopiston digitaalinen arkisto

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