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Baylisascaris potosis n. sp., a new ascarid nematode isolated from captive kinkajou, Potos flavus, from the Cooperative Republic of Guyana
Parasitology International 63 (2014) 591–596
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Parasitology International journal homepage: www.elsevier.com/locate/parint
Baylisascaris potosis n. sp., a new ascarid nematode isolated from captive kinkajou, Potos flavus, from the Cooperative Republic of Guyana Toshihiro Tokiwa a, Shohei Nakamura b, Kensuke Taira b, Yumi Une a,⁎ a b
Laboratory of Pathology, School of Veterinary Medicine, Azabu University, Kanagawa, Japan Laboratory of Parasitology, School of Veterinary Medicine, Azabu University, Kanagawa, Japan
a r t i c l e
i n f o
Article history: Received 7 December 2013 Received in revised form 6 March 2014 Accepted 14 March 2014 Available online 22 March 2014 Keywords: Baylisascaris potosis n. sp. Kinkajou Potos flavus Scanning electron microscopy COX1 28S rDNA
a b s t r a c t We describe a new nematode species, Baylisascaris potosis n. sp., isolated from captive kinkajou, Potos flavus, from the Cooperative Republic of Guyana. The nematode was found in fecal specimens, identified morphologically, and confirmed genetically. The new species is similar to Baylisascaris procyonis, Baylisascaris columnaris, and other Baylisascaris species, but is distinguished by the position of the male phasmidial pole. Nuclear and mitochondrial DNA sequence analyses confirmed that the new species is phylogenetically distinct from all the members of the genus Baylisascaris, and groups with B. procyonis and B. columnaris. This nematode is the 10th species assigned to the genus Baylisascaris. © 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Baylisascaris Sprent 1968 (Nematoda: Ascarididae) is a small genus with only nine recognized species; namely, Baylisascaris transfuga (Rudolphi, 1819), Baylisascaris laevis (Leidy, 1856), Baylisascaris columnaris (Leidy, 1856), Baylisascaris melis (Gedoelst, 1920), Baylisascaris schroederi (McIntosh, 1939), Baylisascaris procyonis (Stefanski and Zarnowski, 1951), Baylisascaris devosi (Sprent, 1952), Baylisascaris ailuri (Wu et al., 1987), and Baylisascaris tasmaniensis Sprent, 1970 [1,2]. Members of the genus Baylisascaris occur primarily in placental carnivores, with B. tasmaniensis and B. laevis occurring in marsupial carnivores and rodents, respectively. Although the raccoon roundworm B. procyonis is recognized as a cause of serious or fatal larva migrans in humans and animals [3,4], other species of the genus Baylisascaris are considered potential zoonotic nematodes [5]. The kinkajou, Potos flavus (Chordata: Procyonidae), is a mediumsized (1.4–4.5 kg), nocturnal, and arboreal mammal native in the lowland rainforests of South and Central America [6]. Previous studies have shown kinkajous to be a definitive host for B. procyonis, the raccoon roundworm [7–9], and morphology of the nematodes identified to B. procyonis isolated from kinkajous in Colombia was reported by Overstreet [7]. Classification of species of the Baylisascaris and the phylogenetic relationships among members of the genus have been proposed based on morphological characters [1,10]. Recently, molecular ⁎ Corresponding author. Tel./fax: +81 42 769 1628. E-mail address: [email protected] (Y. Une).
http://dx.doi.org/10.1016/j.parint.2014.03.003 1383-5769/© 2014 Elsevier Ireland Ltd. All rights reserved.
studies have demonstrated that the second internal transcribed spacer (ITS2), 28S nuclear ribosomal DNA (rDNA), and mitochondrial cytochrome oxidase subunit I (COX1) can provide genetic markers for species-level identification of the genus Baylisascaris [11–13]. However, phylogenetic analyses using the ribosomal ITS2 region indicated that Baylisascaris sp. from a captive kinkajou were different from B. procyonis from raccoons, in spite of morphological similarities [8]. These data suggest that nematodes from kinkajous should be considered to be cryptic species of the genus Baylisascaris, being only distinguished by molecular based analyses. In 2013, we collected fecal samples from two captive kinkajous and found Baylisascaris eggs and nematodes. Considering the importance of B. procyonis and other members of the genus in public health, taxonomic position of Baylisascaris from kinkajou should be revealed. In this study, we characterized nematodes of the genus Baylisascaris collected from two captive kinkajous by morphology and molecular based analyses and described Baylisascaris potosis n. sp. 2. Materials and methods 2.1. Collection of materials The wild female kinkajous, more than two years old, born in the Cooperative Republic of Guyana were imported to Japan in December 2012. Nematodes and eggs were obtained from fresh fecal samples from these kinkajous. Specimens were washed in phosphate buffered saline (pH 7.2), fixed in 10% (v/v) neutral-buffered formalin solution
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T. Tokiwa et al. / Parasitology International 63 (2014) 591–596
and stored in the dark for later study. A body segment was preserved in 100% ethanol and stored at −30 °C until used for molecular analyses. Type specimens were deposited in the National Museum of Nature and Science, Tokyo, Japan.
reaction consisted of 25 cycles each at 96 °C for 10 s, 50 °C for 5 s, and 60 °C for 4 min. Sequence data obtained in this study are available in DDBJ under the accession No. AB901104 for ITS2, No. AB893608 for 28S rDNA and AB893609 for COX1.
2.2. Morphology and morphometric analyses
2.4. DNA sequence analyses
Nematodes were cleared with lactophenol solution (25% glycerin, 25% lactic acid, 25% phenol, and 25% distilled water) in a petri dish, observed using a binocular stereo microscope (Nikon, Japan), and illustrated by the aid of drawing tube fitted to an eyepiece. Eggs were observed by using a Nikon Eclipse E600 microscope (Nikon, Japan). The esophago-intestinal region of worm was fixed in 10% neutral-buffered formalin, embedded in paraffin, sectioned transversally, and stained with hematoxylin and eosin (HE). Measurements were taken with an ocular micrometer and represented in mm as a mean followed by the ranges in parentheses. For scanning electron microscopy, formalinfixed samples were washed three times with distilled water and postfixed in 2% (w/v) osmium tetraxide aqueous solution for 2 h at room temperature. After three washes with phosphate buffer (pH 7.2), the samples were dehydrated twice through an ethanol series (50–99%) and three times in 100%, keeping for 30 min in each concentration. The samples were then immersed in t-butyl alcohol (Wako, Japan) for 30 min at − 30 °C. The samples were freeze-dried using a JFD-310 freeze dryer (JEOL, Japan), sputter-coated with platinum–palladium in a JFC1600 sputter–coater (JEOL), and observed using a JSM-6380-LV scanning electron microscope (JEOL) at the Department of Veterinary Parasitology of the Nippon Veterinary and Life Science University.
Sequence similarity was determined using BLASTN from the National Center for Biotechnology Information website (http://www.ncbi.nlm.nih. gov/Blast.cgi). Newly obtained sequences were used in the phylogenetic analyses. Sequences obtained from GenBank/DDBJ/EMBL databases are as follows: B. procyonis (28S, KC543470, AY821774, U94753; COX1, JF951366), B. columnaris (28S, KC543466-9; COX1, KC543472-5), B. schroederi (28S, JN257013; COX1, HQ671081, EU628682), B. transfuga (28S, KC543471, JN257008-11; COX1, EU628683, EU628684, EU740387, HQ671079, KC543477), B. ailuri (28S, JN257012; COX1, HQ671080), Ascaris lumbricoides (28S, AY210806; COX1, HQ704900), and Toxascaris leonine (28S, JN257000; COX1, KC293927). Sequences were aligned using a web-based version of multiple alignment program (MAFFT, version 7) (http://mafft.cbrc.jp/ alignment/server) [15] with the Q-INS-i setting, followed by manual adjustment using MacClade software, version 4.08 [16]. Analyses were performed using MEGA version 5.2.2 [17]. All positions with gaps or missing data were eliminated from the dataset. The amino acid sequences were translated using invertebrate mitochondrial code (http://www.ncbi.nlm.nih.gov/Taxonomy/Utils/wprintgc.cgi?mode= c#SG5). Uncorrected p-distances were calculated for sequence pairs after removal of insertions and deletions in 2000 bootstrap replicates. Phylogenetic trees were constructed using neighbor-joining (NJ) and maximum likelihood (ML) methods. The best-fit model was estimated using the Akaike Information Criterion (AIC) and the determined Kimura two-parameter plus the gamma distribution of variable sites (G = 0.05) for 28S and Hasegawa–Kishino–Yano plus the gamma distribution of variable sites (G = 0.6) for COX1. Random addition of sequences and stepwise addition of starting trees were used in MP heuristic tree search analysis. Phylogenetic trees were evaluated using the bootstrap methodology based on 2000 replicates for NJ and 1000 replicates for ML.
2.3. Molecular analyses Total DNA was extracted from female nematodes using the NucleoSpin Tissue Kit (TaKaRa-Bio, Japan) according to the manufacturer's protocol. Partial fragments of ITS2, 28S and COX1 were amplified and sequenced. The ITS2 was amplified using primers LC1 and HC2, which corresponded to the conserver 3′ and 5′ ends of the 5.8S-ITS2-28S regions [14]. Primers 28SrDNAF and 28SrDNAR described by Franssen [13] were used to amplify the 28S fragment. The COX1 fragment was amplified using primers BP9443COIF and BP9926COIR, which were designed based on COX1 sequence alignments of B. procyonis (JF951366), B. transfuga (HQ671079), B. schroederi (HQ671081), and B. ailuri (HQ671080). Primer sequences are shown in Table 1. Polymerase chain reaction (PCR) was carried out in 20 μl volumes containing 10× PCR buffer, 25 mM MgCl2, 2.5 mM dNTPs, 50 μM each of the primers, 5 U/μl of TaKaRa Ex Taq polymerase (TaKaRa-Bio), sterilized distilled water, and 1 μl of total DNA. The reaction mixture was first heated at 94 °C for 2 min, followed by 30 cycles at 40 s at 94 °C, 40 s at 60 °C, 1 min at 72 °C, and ending with a final extension at 72 °C for 3 min. PCR products were purified using the Illustra ExoProStar (GE Healthcare Life Science, USA) according to the manufacture's protocol. The purified PCR products were then sequenced in the ABI Genetic Analyzer (Applied Biosystems, USA) using the Big Dye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, USA). The same primers for amplification described above were used for the forward and reverse sequencing in two separate reactions. The
3. Results 3.1. Description of the new species 3.1.1. Baylisascaris potosis n. sp. (Figs. 1, 2) Family: Ascarididae Baird, 1853 Subfamily: Ascaridinae (Baird, 1853) Genus: Baylisascaris Sprent, 1968 General: Body with a thick cuticle, cylindrical, slightly tapered at both ends. Ridges of three well-defined lips detigerous (Figs. 1a, b, 2a). Denticles sharp, equilateral triangle (Figs. 1b, 2a). The dorsal lip with double dorso-lateral papillae (Figs. 1b, 2a, b); the subventral lips with one double ventro-lateral and one lateral papillae and one amphid (Figs. 1b, 2a, c). Cervical alae inconspicuous but cuticular bars reaching the surface of cuticle in lateral field at base of esophago-intestinal junction (Fig. 1c).
Table 1 Primers used in this study for PCR amplification and sequencing. Primers
Amplified regions
Direction
PCR primers
Reference
LC1 HC2 BP9443COIF BP9926COIR 28SrDNAF 28SrDNAR
ITS2 ITS2 COX1 COX1 28S 28S
F R F R F R
5′-CGACTATCGATGAAGAACGCAGC-3′ 5′-ATATGCTTAAGTTCAGCGGG-3′ 5′-TTTTTCCTCATCCTGAGGTTT-3′ 5′-CTCCACCATAAAGTCACACCAG-3′ 5′-CGAGGATTCCCTTAGTAACT-3′ 5′-TCGGATAGGTGGTCAACG-3′
[14] [14] In this study In this study [9] [9]
F, forward primer; R, reverse primer.
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Fig. 1. Baylisascaris potosis n. sp., adult male. a — Dorsal view of anterior body. b — Apical view of lips. c — Cuticle in lateral field at base of esophageal–intestinal junction. d — Tail. e — Lateral view of spicules. f — Unembryonated egg showing thick surface (upper right). Scales apply to all figures to the right.
Male (based on holotype and two males): Body 121 (117–127) long by 1.14 (1.10–1.21) wide at esophagus level, 1.66 (1.60–1.75) wide at midbody. Esophagus 4.25 (4.10–4.40) long by 0.44 (0.35–0.50) wide. Spicles 0.66 (0.61–0.72) long (Fig. 1e). Pre- and post-cloacal areas rugose with round anterior and posterior margins, respectively (Figs. 1d, 2e, f). Pre-cloacal papillae counts averaged 47.3 (44–52), situated in slightly divergent rows with somewhat irregular space (Figs. 1d, 2e). A central papilla situated on the anterior part of pre-cloacal area (Figs. 1d, 2e). Post-cloacal papillae five pairs: the first and second pairs double; the third and fourth pairs single; the fifth pair phasmid located on the sub-ventral side of the fourth pair (Figs. 1d, 2e). In a single male, the second post-cloacal papillae not fused and appeared as two single papillae (Fig. 2e). Tail relatively long, 0.56 (0.54–0.60) with a mucronate termination (Figs. 1d, 2e). Female (based on allotype and a female, otherwise stated): Body 219 (214–223) long by 1.65 (1.60–1.71) wide at esophagus level, 2.71 (2.51–2.90) at midbody. Esophagus 4.56 long by 0.44 wide. Vulva opened 57 (28.2% of body length) from the tail end. Tail 1.15 (1.10– 1.20). Phasmids on the sub-ventral side, less than one third distance between tail end and anus (Fig. 2d).
Eggs (based on 30 specimens): Unembryonated eggs, 0.087 (0.079– 0.094) long by 0.071 (0.064–0.078) wide; embryonated eggs, 0.083 (0.073–0.095) long by 0.073 (0.069–0.078) wide. Egg shells finely pitted (Figs. 1f, 2g). 3.1.2. Taxonomic summary Type host: Potos flavus (Schreber, 1774) (Carnivora: Procyonidae) Type locality: The Cooperative Republic of Guyana Type materials: Holotype, adult male (NSMT-As3965); allotype, adult female (NSMT-As3966); paratype, adult male (NSMT-As3967). Etymology: The new species is named for the type host 3.1.3. Remarks Present new species is assigned to the genus Baylisascaris based on the presence of rough areas in the male peri-cloacal regions, both in front of and behind the cloaca. B. potosis n. sp. has inconspicuous cervical alae with cuticular bars that reach the cuticle surface. These characteristics separate B. potosis n. sp. from B. transfuga, B. melis, B. ailuri, and B. tasmaniensis having salient cervical alae [1,10,18–20]. B. potosis n. sp. differs from B. schroederi by the number of precloacal papillae
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Fig. 2. Scanning electron microscopy of B. potosis n. sp. a — Apical view of male head. b — Dorsal view of dorsal lip of male. c — Dorsal view of subventral lip of male. d — Ventral view of female posterior part. e — Ventral view of male posterior part. f — Posterior part of rugose pericloacal area. g — egg. Scale bars: a, e = 100 μm, b, c = 50 μm, d = 200 μm, f, g = 20 μm. Abbreviations: DL, dorsal lip; SVL, subventral lip; DLP, double lateral papillae; AM, amphidial pore; LP, lateral papillae; DVP, double ventral papillae; A, anus; PH, phasmidial pore; PCP, Precloacal central papillae; SP, spicule.
(44–52 vs. 65–70) and inconspicuous denticles in B. schroederi [1,10,11]. B. potosis n. sp. differs from B. devosi and B. laevis in the shape of the posterior margin of the rugose pericloacal area [21,22]. Male B. potosis n. sp. has phasmids at the most posterior end of the postcloacal papillae. These characteristics separate B. potosis n. sp. from B. procyonis and B. columnaris, which have phasmid located between and somewhat medial to the two postcloacal papillae [23–27]. Overstreet first described B. procyonis obtained from kinkajous in Colombia [7]. He recognized marked differences between B. procyonis from kinkajou and those from raccoons, such as the morphology of the posterior margin of the rugose pericloacal area and phasmid location; however, he considered that these differences were not sufficiently quantified to differentiate species. The three B. potosis males used in our description possessed a round posterior margin in rugose postcloacal area. On the other hand, the single degenerated male from kinkajous showed pointed posterior margins (data not shown). Presumably, the morphological change observed by Overstreet and the authors in the single male might be due to a postmortal degradation. 3.2. Phylogenetic analyses ITS2 was obtained from a female B. potosis n. sp. The 434-bp amplicon had 100% identity with the sequence of Baylisascaris sp. (KF680774) isolated from kinkajou in our previous study [8]. 28S rDNA was obtained from a female B. potosis n. sp. The 702-bp amplicon was 48.7% AT. BLAST analysis showed 99% identity with B. columnaris (KC543466-9) and B. procyonis (KC543470, U94753, AY821774). Compared to other Baylisascaris species available in the GenBank/DDBJ/EMBL databases, there were a total of 26 variable
positions in the 28S sequence (18 parsimony-informative and 8 singleton sites) in the 702-bp pairwise alignment. Interspecific p-distance between B. potosis n. sp. and other species ranged from 0.012 (B. columnaris) to 0.025 (B. transfuga and B. ailuri). COX1 sequences were obtained from two B. potosis n. sp. specimens (female and male). There were no substitutions between these two sequences. The length and AT content of COX1 sequences were 406 bp and 67.0%, respectively. BLAST analysis showed that B. potosis shared 95% identity with previously reported COX1 sequences from B. columnaris (KC543472-5) and B. procyonis (JF951366). When comparing the COX1 sequence of B. potosis n. sp. to the other Baylisascaris species available in GenBank/DDBJ/EMBL databases, a total of 50 variable positions (34 parsimony-informative and 16 singleton sites) appeared in the 402-bp pairwise alignment. The amino acid sequence alignment was 134 aa long. B. potosis and P. procyonis differed at 17 amino acids, while B. potosis and B. columnaris differed at 19 amino acids. Interspecific p-distance between B. potosis n. sp. and other Baylisascaris species ranged from 0.058 (B. columnaris and B. procyonis) to 0.096 (B. schroederi). Phylogenetic trees based on 28S and COX1 partial sequences were obtained by comparing sequences of B. potosis n. sp. and selected Baylisascaris nematode sequences in the GenBank/DDBJ/EMBL databases. Phylogenetic analyses using NJ and ML showed similar tree topology (Fig. 3). Species of Baylisascaris assembled in a well-supported monophyletic clade. Phylogenetic trees constructed using 28S and COX1 partial sequences confirmed B. potosis n. sp. to be within the well-supported clade containing B. procyonis and B. columnaris. The results obtained from phylogenetic analyses support that B. potosis n. sp. is a distinct species.
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Fig. 3. Phylogenetic tree based on 28S rDNA (a) and COX1 (b) sequences of Baylisascaris spp., showing the position of B. potosis n. sp. inferred by NJ and ML. Numbers at nodes represent bootstrap values (NJ/ML).
4. Discussion According to Sprent (1968, 1970) [1,10], Baylisascaris species are morphologically divided into three groups. The first, group 1, contains B. transfuga, B. melis, and B. tasmaniensis; members of this group are larger and with conspicuous cervical alae. B. ailuri, with conspicuous cervical alae, would also be included in this group. Group 2 contains B. procyonis, B. columnaris, B. devosi, and B. laevis; these members are smaller, with inconspicuous cervical alae. Finally, Group 3 is comprised of only B. schroederi, where the female has a longer tail and the male more postcloacal papillae; the cervical alae are also inconspicuous. Based on these morphological characteristics B. potosis n. sp. belongs to Group 2. Although the morphological characteristics of B. potosis n. sp. do not generally distinguish it from B. procyonis and B. columnaris, important diagnostic characteristics, such as male phasmid location, were identified. In order to overcome the inadequacy of morphological analyses for reliable differentiation of this new Baylisascaris species, a major objective of this study was the development of molecular tools for reliable identification of B. potosis n. sp. Phylogenetic trees based on different approaches (NJ and ML) indicated two major Baylisascaris clades with high
bootstrap support. The 28S tree showed a close relationship between B. schroederi and B. ailuri and than to B. transfuga. However, in the COX1 tree, B. ailuri was more closely related to B. transfuga than to B. schroederi. The COX1 tree was consistent with a recent study of 12S rDNA [12] and amino acid sequences from 12 mitochondrial genes [2,28]. In the present study, both trees demonstrate that B. potosis was more closely related to B. procyonis and B. columnaris than to other Baylisascaris species. Given that B. potosis isolated from kinkajous is genetically and morphologically closely related to B. procyonis, there exists a potential risk for human infection with B. potosis. Acknowledgments The authors would like to thank the anonymous reviewers for their valuable comments and suggestions to improve the quality of the paper. We also thank Department of Veterinary Parasitology, Nippon Veterinary and Life Science University for use of SEM. This work was supported by a Grant-in-Aid for Research on Emerging and Re-emerging Infectious Diseases from the Ministry of Health, Labor and Welfare of Japan (H24-Shinko-Ippan-006).
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Parasitology International journal homepage: www.elsevier.com/locate/parint
Baylisascaris potosis n. sp., a new ascarid nematode isolated from captive kinkajou, Potos flavus, from the Cooperative Republic of Guyana Toshihiro Tokiwa a, Shohei Nakamura b, Kensuke Taira b, Yumi Une a,⁎ a b
Laboratory of Pathology, School of Veterinary Medicine, Azabu University, Kanagawa, Japan Laboratory of Parasitology, School of Veterinary Medicine, Azabu University, Kanagawa, Japan
a r t i c l e
i n f o
Article history: Received 7 December 2013 Received in revised form 6 March 2014 Accepted 14 March 2014 Available online 22 March 2014 Keywords: Baylisascaris potosis n. sp. Kinkajou Potos flavus Scanning electron microscopy COX1 28S rDNA
a b s t r a c t We describe a new nematode species, Baylisascaris potosis n. sp., isolated from captive kinkajou, Potos flavus, from the Cooperative Republic of Guyana. The nematode was found in fecal specimens, identified morphologically, and confirmed genetically. The new species is similar to Baylisascaris procyonis, Baylisascaris columnaris, and other Baylisascaris species, but is distinguished by the position of the male phasmidial pole. Nuclear and mitochondrial DNA sequence analyses confirmed that the new species is phylogenetically distinct from all the members of the genus Baylisascaris, and groups with B. procyonis and B. columnaris. This nematode is the 10th species assigned to the genus Baylisascaris. © 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Baylisascaris Sprent 1968 (Nematoda: Ascarididae) is a small genus with only nine recognized species; namely, Baylisascaris transfuga (Rudolphi, 1819), Baylisascaris laevis (Leidy, 1856), Baylisascaris columnaris (Leidy, 1856), Baylisascaris melis (Gedoelst, 1920), Baylisascaris schroederi (McIntosh, 1939), Baylisascaris procyonis (Stefanski and Zarnowski, 1951), Baylisascaris devosi (Sprent, 1952), Baylisascaris ailuri (Wu et al., 1987), and Baylisascaris tasmaniensis Sprent, 1970 [1,2]. Members of the genus Baylisascaris occur primarily in placental carnivores, with B. tasmaniensis and B. laevis occurring in marsupial carnivores and rodents, respectively. Although the raccoon roundworm B. procyonis is recognized as a cause of serious or fatal larva migrans in humans and animals [3,4], other species of the genus Baylisascaris are considered potential zoonotic nematodes [5]. The kinkajou, Potos flavus (Chordata: Procyonidae), is a mediumsized (1.4–4.5 kg), nocturnal, and arboreal mammal native in the lowland rainforests of South and Central America [6]. Previous studies have shown kinkajous to be a definitive host for B. procyonis, the raccoon roundworm [7–9], and morphology of the nematodes identified to B. procyonis isolated from kinkajous in Colombia was reported by Overstreet [7]. Classification of species of the Baylisascaris and the phylogenetic relationships among members of the genus have been proposed based on morphological characters [1,10]. Recently, molecular ⁎ Corresponding author. Tel./fax: +81 42 769 1628. E-mail address: [email protected] (Y. Une).
http://dx.doi.org/10.1016/j.parint.2014.03.003 1383-5769/© 2014 Elsevier Ireland Ltd. All rights reserved.
studies have demonstrated that the second internal transcribed spacer (ITS2), 28S nuclear ribosomal DNA (rDNA), and mitochondrial cytochrome oxidase subunit I (COX1) can provide genetic markers for species-level identification of the genus Baylisascaris [11–13]. However, phylogenetic analyses using the ribosomal ITS2 region indicated that Baylisascaris sp. from a captive kinkajou were different from B. procyonis from raccoons, in spite of morphological similarities [8]. These data suggest that nematodes from kinkajous should be considered to be cryptic species of the genus Baylisascaris, being only distinguished by molecular based analyses. In 2013, we collected fecal samples from two captive kinkajous and found Baylisascaris eggs and nematodes. Considering the importance of B. procyonis and other members of the genus in public health, taxonomic position of Baylisascaris from kinkajou should be revealed. In this study, we characterized nematodes of the genus Baylisascaris collected from two captive kinkajous by morphology and molecular based analyses and described Baylisascaris potosis n. sp. 2. Materials and methods 2.1. Collection of materials The wild female kinkajous, more than two years old, born in the Cooperative Republic of Guyana were imported to Japan in December 2012. Nematodes and eggs were obtained from fresh fecal samples from these kinkajous. Specimens were washed in phosphate buffered saline (pH 7.2), fixed in 10% (v/v) neutral-buffered formalin solution
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and stored in the dark for later study. A body segment was preserved in 100% ethanol and stored at −30 °C until used for molecular analyses. Type specimens were deposited in the National Museum of Nature and Science, Tokyo, Japan.
reaction consisted of 25 cycles each at 96 °C for 10 s, 50 °C for 5 s, and 60 °C for 4 min. Sequence data obtained in this study are available in DDBJ under the accession No. AB901104 for ITS2, No. AB893608 for 28S rDNA and AB893609 for COX1.
2.2. Morphology and morphometric analyses
2.4. DNA sequence analyses
Nematodes were cleared with lactophenol solution (25% glycerin, 25% lactic acid, 25% phenol, and 25% distilled water) in a petri dish, observed using a binocular stereo microscope (Nikon, Japan), and illustrated by the aid of drawing tube fitted to an eyepiece. Eggs were observed by using a Nikon Eclipse E600 microscope (Nikon, Japan). The esophago-intestinal region of worm was fixed in 10% neutral-buffered formalin, embedded in paraffin, sectioned transversally, and stained with hematoxylin and eosin (HE). Measurements were taken with an ocular micrometer and represented in mm as a mean followed by the ranges in parentheses. For scanning electron microscopy, formalinfixed samples were washed three times with distilled water and postfixed in 2% (w/v) osmium tetraxide aqueous solution for 2 h at room temperature. After three washes with phosphate buffer (pH 7.2), the samples were dehydrated twice through an ethanol series (50–99%) and three times in 100%, keeping for 30 min in each concentration. The samples were then immersed in t-butyl alcohol (Wako, Japan) for 30 min at − 30 °C. The samples were freeze-dried using a JFD-310 freeze dryer (JEOL, Japan), sputter-coated with platinum–palladium in a JFC1600 sputter–coater (JEOL), and observed using a JSM-6380-LV scanning electron microscope (JEOL) at the Department of Veterinary Parasitology of the Nippon Veterinary and Life Science University.
Sequence similarity was determined using BLASTN from the National Center for Biotechnology Information website (http://www.ncbi.nlm.nih. gov/Blast.cgi). Newly obtained sequences were used in the phylogenetic analyses. Sequences obtained from GenBank/DDBJ/EMBL databases are as follows: B. procyonis (28S, KC543470, AY821774, U94753; COX1, JF951366), B. columnaris (28S, KC543466-9; COX1, KC543472-5), B. schroederi (28S, JN257013; COX1, HQ671081, EU628682), B. transfuga (28S, KC543471, JN257008-11; COX1, EU628683, EU628684, EU740387, HQ671079, KC543477), B. ailuri (28S, JN257012; COX1, HQ671080), Ascaris lumbricoides (28S, AY210806; COX1, HQ704900), and Toxascaris leonine (28S, JN257000; COX1, KC293927). Sequences were aligned using a web-based version of multiple alignment program (MAFFT, version 7) (http://mafft.cbrc.jp/ alignment/server) [15] with the Q-INS-i setting, followed by manual adjustment using MacClade software, version 4.08 [16]. Analyses were performed using MEGA version 5.2.2 [17]. All positions with gaps or missing data were eliminated from the dataset. The amino acid sequences were translated using invertebrate mitochondrial code (http://www.ncbi.nlm.nih.gov/Taxonomy/Utils/wprintgc.cgi?mode= c#SG5). Uncorrected p-distances were calculated for sequence pairs after removal of insertions and deletions in 2000 bootstrap replicates. Phylogenetic trees were constructed using neighbor-joining (NJ) and maximum likelihood (ML) methods. The best-fit model was estimated using the Akaike Information Criterion (AIC) and the determined Kimura two-parameter plus the gamma distribution of variable sites (G = 0.05) for 28S and Hasegawa–Kishino–Yano plus the gamma distribution of variable sites (G = 0.6) for COX1. Random addition of sequences and stepwise addition of starting trees were used in MP heuristic tree search analysis. Phylogenetic trees were evaluated using the bootstrap methodology based on 2000 replicates for NJ and 1000 replicates for ML.
2.3. Molecular analyses Total DNA was extracted from female nematodes using the NucleoSpin Tissue Kit (TaKaRa-Bio, Japan) according to the manufacturer's protocol. Partial fragments of ITS2, 28S and COX1 were amplified and sequenced. The ITS2 was amplified using primers LC1 and HC2, which corresponded to the conserver 3′ and 5′ ends of the 5.8S-ITS2-28S regions [14]. Primers 28SrDNAF and 28SrDNAR described by Franssen [13] were used to amplify the 28S fragment. The COX1 fragment was amplified using primers BP9443COIF and BP9926COIR, which were designed based on COX1 sequence alignments of B. procyonis (JF951366), B. transfuga (HQ671079), B. schroederi (HQ671081), and B. ailuri (HQ671080). Primer sequences are shown in Table 1. Polymerase chain reaction (PCR) was carried out in 20 μl volumes containing 10× PCR buffer, 25 mM MgCl2, 2.5 mM dNTPs, 50 μM each of the primers, 5 U/μl of TaKaRa Ex Taq polymerase (TaKaRa-Bio), sterilized distilled water, and 1 μl of total DNA. The reaction mixture was first heated at 94 °C for 2 min, followed by 30 cycles at 40 s at 94 °C, 40 s at 60 °C, 1 min at 72 °C, and ending with a final extension at 72 °C for 3 min. PCR products were purified using the Illustra ExoProStar (GE Healthcare Life Science, USA) according to the manufacture's protocol. The purified PCR products were then sequenced in the ABI Genetic Analyzer (Applied Biosystems, USA) using the Big Dye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, USA). The same primers for amplification described above were used for the forward and reverse sequencing in two separate reactions. The
3. Results 3.1. Description of the new species 3.1.1. Baylisascaris potosis n. sp. (Figs. 1, 2) Family: Ascarididae Baird, 1853 Subfamily: Ascaridinae (Baird, 1853) Genus: Baylisascaris Sprent, 1968 General: Body with a thick cuticle, cylindrical, slightly tapered at both ends. Ridges of three well-defined lips detigerous (Figs. 1a, b, 2a). Denticles sharp, equilateral triangle (Figs. 1b, 2a). The dorsal lip with double dorso-lateral papillae (Figs. 1b, 2a, b); the subventral lips with one double ventro-lateral and one lateral papillae and one amphid (Figs. 1b, 2a, c). Cervical alae inconspicuous but cuticular bars reaching the surface of cuticle in lateral field at base of esophago-intestinal junction (Fig. 1c).
Table 1 Primers used in this study for PCR amplification and sequencing. Primers
Amplified regions
Direction
PCR primers
Reference
LC1 HC2 BP9443COIF BP9926COIR 28SrDNAF 28SrDNAR
ITS2 ITS2 COX1 COX1 28S 28S
F R F R F R
5′-CGACTATCGATGAAGAACGCAGC-3′ 5′-ATATGCTTAAGTTCAGCGGG-3′ 5′-TTTTTCCTCATCCTGAGGTTT-3′ 5′-CTCCACCATAAAGTCACACCAG-3′ 5′-CGAGGATTCCCTTAGTAACT-3′ 5′-TCGGATAGGTGGTCAACG-3′
[14] [14] In this study In this study [9] [9]
F, forward primer; R, reverse primer.
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Fig. 1. Baylisascaris potosis n. sp., adult male. a — Dorsal view of anterior body. b — Apical view of lips. c — Cuticle in lateral field at base of esophageal–intestinal junction. d — Tail. e — Lateral view of spicules. f — Unembryonated egg showing thick surface (upper right). Scales apply to all figures to the right.
Male (based on holotype and two males): Body 121 (117–127) long by 1.14 (1.10–1.21) wide at esophagus level, 1.66 (1.60–1.75) wide at midbody. Esophagus 4.25 (4.10–4.40) long by 0.44 (0.35–0.50) wide. Spicles 0.66 (0.61–0.72) long (Fig. 1e). Pre- and post-cloacal areas rugose with round anterior and posterior margins, respectively (Figs. 1d, 2e, f). Pre-cloacal papillae counts averaged 47.3 (44–52), situated in slightly divergent rows with somewhat irregular space (Figs. 1d, 2e). A central papilla situated on the anterior part of pre-cloacal area (Figs. 1d, 2e). Post-cloacal papillae five pairs: the first and second pairs double; the third and fourth pairs single; the fifth pair phasmid located on the sub-ventral side of the fourth pair (Figs. 1d, 2e). In a single male, the second post-cloacal papillae not fused and appeared as two single papillae (Fig. 2e). Tail relatively long, 0.56 (0.54–0.60) with a mucronate termination (Figs. 1d, 2e). Female (based on allotype and a female, otherwise stated): Body 219 (214–223) long by 1.65 (1.60–1.71) wide at esophagus level, 2.71 (2.51–2.90) at midbody. Esophagus 4.56 long by 0.44 wide. Vulva opened 57 (28.2% of body length) from the tail end. Tail 1.15 (1.10– 1.20). Phasmids on the sub-ventral side, less than one third distance between tail end and anus (Fig. 2d).
Eggs (based on 30 specimens): Unembryonated eggs, 0.087 (0.079– 0.094) long by 0.071 (0.064–0.078) wide; embryonated eggs, 0.083 (0.073–0.095) long by 0.073 (0.069–0.078) wide. Egg shells finely pitted (Figs. 1f, 2g). 3.1.2. Taxonomic summary Type host: Potos flavus (Schreber, 1774) (Carnivora: Procyonidae) Type locality: The Cooperative Republic of Guyana Type materials: Holotype, adult male (NSMT-As3965); allotype, adult female (NSMT-As3966); paratype, adult male (NSMT-As3967). Etymology: The new species is named for the type host 3.1.3. Remarks Present new species is assigned to the genus Baylisascaris based on the presence of rough areas in the male peri-cloacal regions, both in front of and behind the cloaca. B. potosis n. sp. has inconspicuous cervical alae with cuticular bars that reach the cuticle surface. These characteristics separate B. potosis n. sp. from B. transfuga, B. melis, B. ailuri, and B. tasmaniensis having salient cervical alae [1,10,18–20]. B. potosis n. sp. differs from B. schroederi by the number of precloacal papillae
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Fig. 2. Scanning electron microscopy of B. potosis n. sp. a — Apical view of male head. b — Dorsal view of dorsal lip of male. c — Dorsal view of subventral lip of male. d — Ventral view of female posterior part. e — Ventral view of male posterior part. f — Posterior part of rugose pericloacal area. g — egg. Scale bars: a, e = 100 μm, b, c = 50 μm, d = 200 μm, f, g = 20 μm. Abbreviations: DL, dorsal lip; SVL, subventral lip; DLP, double lateral papillae; AM, amphidial pore; LP, lateral papillae; DVP, double ventral papillae; A, anus; PH, phasmidial pore; PCP, Precloacal central papillae; SP, spicule.
(44–52 vs. 65–70) and inconspicuous denticles in B. schroederi [1,10,11]. B. potosis n. sp. differs from B. devosi and B. laevis in the shape of the posterior margin of the rugose pericloacal area [21,22]. Male B. potosis n. sp. has phasmids at the most posterior end of the postcloacal papillae. These characteristics separate B. potosis n. sp. from B. procyonis and B. columnaris, which have phasmid located between and somewhat medial to the two postcloacal papillae [23–27]. Overstreet first described B. procyonis obtained from kinkajous in Colombia [7]. He recognized marked differences between B. procyonis from kinkajou and those from raccoons, such as the morphology of the posterior margin of the rugose pericloacal area and phasmid location; however, he considered that these differences were not sufficiently quantified to differentiate species. The three B. potosis males used in our description possessed a round posterior margin in rugose postcloacal area. On the other hand, the single degenerated male from kinkajous showed pointed posterior margins (data not shown). Presumably, the morphological change observed by Overstreet and the authors in the single male might be due to a postmortal degradation. 3.2. Phylogenetic analyses ITS2 was obtained from a female B. potosis n. sp. The 434-bp amplicon had 100% identity with the sequence of Baylisascaris sp. (KF680774) isolated from kinkajou in our previous study [8]. 28S rDNA was obtained from a female B. potosis n. sp. The 702-bp amplicon was 48.7% AT. BLAST analysis showed 99% identity with B. columnaris (KC543466-9) and B. procyonis (KC543470, U94753, AY821774). Compared to other Baylisascaris species available in the GenBank/DDBJ/EMBL databases, there were a total of 26 variable
positions in the 28S sequence (18 parsimony-informative and 8 singleton sites) in the 702-bp pairwise alignment. Interspecific p-distance between B. potosis n. sp. and other species ranged from 0.012 (B. columnaris) to 0.025 (B. transfuga and B. ailuri). COX1 sequences were obtained from two B. potosis n. sp. specimens (female and male). There were no substitutions between these two sequences. The length and AT content of COX1 sequences were 406 bp and 67.0%, respectively. BLAST analysis showed that B. potosis shared 95% identity with previously reported COX1 sequences from B. columnaris (KC543472-5) and B. procyonis (JF951366). When comparing the COX1 sequence of B. potosis n. sp. to the other Baylisascaris species available in GenBank/DDBJ/EMBL databases, a total of 50 variable positions (34 parsimony-informative and 16 singleton sites) appeared in the 402-bp pairwise alignment. The amino acid sequence alignment was 134 aa long. B. potosis and P. procyonis differed at 17 amino acids, while B. potosis and B. columnaris differed at 19 amino acids. Interspecific p-distance between B. potosis n. sp. and other Baylisascaris species ranged from 0.058 (B. columnaris and B. procyonis) to 0.096 (B. schroederi). Phylogenetic trees based on 28S and COX1 partial sequences were obtained by comparing sequences of B. potosis n. sp. and selected Baylisascaris nematode sequences in the GenBank/DDBJ/EMBL databases. Phylogenetic analyses using NJ and ML showed similar tree topology (Fig. 3). Species of Baylisascaris assembled in a well-supported monophyletic clade. Phylogenetic trees constructed using 28S and COX1 partial sequences confirmed B. potosis n. sp. to be within the well-supported clade containing B. procyonis and B. columnaris. The results obtained from phylogenetic analyses support that B. potosis n. sp. is a distinct species.
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Fig. 3. Phylogenetic tree based on 28S rDNA (a) and COX1 (b) sequences of Baylisascaris spp., showing the position of B. potosis n. sp. inferred by NJ and ML. Numbers at nodes represent bootstrap values (NJ/ML).
4. Discussion According to Sprent (1968, 1970) [1,10], Baylisascaris species are morphologically divided into three groups. The first, group 1, contains B. transfuga, B. melis, and B. tasmaniensis; members of this group are larger and with conspicuous cervical alae. B. ailuri, with conspicuous cervical alae, would also be included in this group. Group 2 contains B. procyonis, B. columnaris, B. devosi, and B. laevis; these members are smaller, with inconspicuous cervical alae. Finally, Group 3 is comprised of only B. schroederi, where the female has a longer tail and the male more postcloacal papillae; the cervical alae are also inconspicuous. Based on these morphological characteristics B. potosis n. sp. belongs to Group 2. Although the morphological characteristics of B. potosis n. sp. do not generally distinguish it from B. procyonis and B. columnaris, important diagnostic characteristics, such as male phasmid location, were identified. In order to overcome the inadequacy of morphological analyses for reliable differentiation of this new Baylisascaris species, a major objective of this study was the development of molecular tools for reliable identification of B. potosis n. sp. Phylogenetic trees based on different approaches (NJ and ML) indicated two major Baylisascaris clades with high
bootstrap support. The 28S tree showed a close relationship between B. schroederi and B. ailuri and than to B. transfuga. However, in the COX1 tree, B. ailuri was more closely related to B. transfuga than to B. schroederi. The COX1 tree was consistent with a recent study of 12S rDNA [12] and amino acid sequences from 12 mitochondrial genes [2,28]. In the present study, both trees demonstrate that B. potosis was more closely related to B. procyonis and B. columnaris than to other Baylisascaris species. Given that B. potosis isolated from kinkajous is genetically and morphologically closely related to B. procyonis, there exists a potential risk for human infection with B. potosis. Acknowledgments The authors would like to thank the anonymous reviewers for their valuable comments and suggestions to improve the quality of the paper. We also thank Department of Veterinary Parasitology, Nippon Veterinary and Life Science University for use of SEM. This work was supported by a Grant-in-Aid for Research on Emerging and Re-emerging Infectious Diseases from the Ministry of Health, Labor and Welfare of Japan (H24-Shinko-Ippan-006).
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