ORIGINAL ARTICLE /ARTÍCULO ORIGINAL

MORPHOLOGICAL AND MOLECULAR DESCRIPTION OF BAYLISASCARIS

VENEZUELENSIS, N. SP. FROM A NATURAL INFECTION IN THE SOUTH AMERICAN

SPECTACLED BEAR TREMARCTOS ORNATUS CUVIER, 1825 IN VENEZUELA

CARACTERIZACIÓN MORFOLÓGICA Y MOLECULAR DE BAYLISASCARIS

VENEZUELENSIS, N. SP. DE UNA INFECCIÓN NATURAL EN EL OSO ANDINO DE

ANTEOJOS, TREMARCTOS ORNATUS CUVIER, 1825 EN VENEZUELA

1* 1 2

Arlett Pérez Mata ; Herakles García Pérez & José Gauta Parra

1 Centro de Investigación en Parasitología Veterinaria “Dr. Manuel Antonio Rivera Acevedo” (CIPV-MARA), Facultad de

Ciencias Veterinarias, Universidad Central de Venezuela, Campus Maracay (FCV-UCV). Maracay 2101, State of Aragua,

Venezuela.

2 Universidad Politécnica Kleber Ramírez, extensión Bailadores, PNF Agrotecnia. Bailadores, State of Mérida, Venezuela.

* Corresponding author. E-mail: arlettperez@gmail.com

Neotropical Helminthology, 2016, 10(1), ene-jun: 85-103.

ABSTRACT

Keywords: Baylisascaris – ITS2 – ITS1 – 5.8S – Tremarctos ornatus – Venezuela

In this study we report the first description of natural infection with Baylisascaris sp. in the South

American spectacled bear (Tremarctos ornatus Cuvier, 1825) from Venezuela. In November

2010, a spectacled bear was found dead during a routine patrol in The National Park “India Carú”

(Bailadores Mérida, Venezuelan Andes). Necropsy revealed congestion, hemorrhage of the lungs

and small bowel enlargement. Large amount of big whitish nematodes were found filling the

intestinal lumen. Nematodes were initially identified as Baylisascaris sp., and they were

established as morphologically closely related to Baylisascaris transfuga (Rudolphi, 1819),

differing in the number of caudal papillae. Baylisascaris transfuga has been reported in several

species of bears in different countries. Molecular characterization based on analysis of the ITS1,

ITS2 rDNA and 5.8S region positioned the spectacled bear specimen in the genus Baylisascaris.

Furthermore, important divergences were noted when compared to homologous sequences from

other members of this genus retrieved from the GenBank. Indeed, several SNPs located into the

highly conserved 5.8S as compared with B. transfuga, B. schroederi (McIntosh, 1939), B.

procyonis (Stefanski & Zarnowski, 1951) and B. columnaris Leidy, 1856, as well as a genealogy

network based on complete ITS rDNA sequences (ITS1+5.8S+ITS2) strongly suggest that this

specimen constitute a new species of Baylisascaris. Taking into account morphological and

molecular evidence, the vertebrate host and its particular geographical distribution in South

America, we describe our specimens as a new species within this genus and we name it

Baylisascaris venezuelensis.

ISSN Versión impresa 2218-6425 ISSN Versión Electrónica 1995-1043

RESUMEN

Palabras clave: Baylisascaris - ITS2 - ITS1 - 5.8S - Tremarctos ornatus - Venezuela

En este estudio describimos por primera vez un caso de infección natural por Baylisascaris sp. en

un oso andino de anteojos (Tremarctos ornatus Cuvier, 1825) en Venezuela. En noviembre de

2010 un joven oso fue encontrado muerto por los Oficiales de la Guardia Nacional durante un

patrullaje rutinario en el parque Nacional India Carú en Bailadores, (Mérida, Andes

Venezolanos). La necropsia reveló congestión y hemorragia de los pulmones y agrandamiento

del volumen del intestino delgado. Se encontró una gran cantidad de nematodos blanquecinos de

gran tamaño llenando el lumen intestinal. Los nematodos fueron morfológicamente identificados

como Baylisascaris sp. morfológicamente cercano a Baylisascaris transfuga (Rudolphi, 1819),

difiriendo en el número de papilas caudales. Baylisascaris transfuga ha sido registrado en

diferentes especies de osos en diferentes países. La caracterización molecular de nuestro

espécimen basado en el análisis del ITS1, ITS2 rDNA y la región del 5.8S lo posiciona dentro del

género Baylisascaris. Adicionalmente, se observaron importantes divergencias cuando se

comparó con secuencias homólogas de otros miembros del género obtenidas del GenBank. De

hecho, varios SNPs localizados dentro de la región altamente conservada 5.8S al compararlo con

B. transfuga, B. schroederi (McIntosh, 1939), B. procyonis (Stefanski & Zarnowski, 1951) y B.

columnaris Leidy, 1856, así como la genealogía basada en la secuencia completa del ITS rDNA

(ITS1+5.8S+ITS2) sugiere fuertemente que este espécimen constituye una nueva especie de

Baylisascaris. Tomando en cuenta la evidencia morfológica y molecular, la especie hospedadora

y su particular y restringida distribución geográfica en los andes suramericanos, sugerimos se

considere a nuestro espécimen una nueva especie con el nombre de Baylisascaris venezuelensis.

Neotropical Helminthology. Vol. 10, Nº1, ene-jun 2016

INTRODUCTION

The South American spectacled bear

Tremarctos ornatus Cuvier, 1825 is the only

species of the family Ursidae inhabiting South

America. Its geographical distribution

comprises the Andean Mountains of

Venezuela, Colombia, Ecuador, Peru and

Bolivia, and the northern borderline of

Argentina. An outstanding morphological

feature is the presence of a kind of mask (white

circle hair marks) surrounding eyes. This white

mask can vary greatly from one animal to other

(Röhl, 1949; Mondolfi, 1971; Goldstein,

1989). Despite the importance of the study of

animal diseases for conservation efforts, there

is a pronounced lack of knowledge about

pathogen diversity and susceptibility in

wildlife (MacPhee & Greenwood, 2013). It is

generally accepted that in their natural habitat

the spectacled bears may be affected by several

infectious agents, mainly during the first stages

of their life (cubs and juvenile). Moreover,

anthropogenic factors leading to a suddenly

habitat loss and fragmentation give little time

for wildlife to adjust to their new

circumstances and may cause severe stress on

individual species. In fact, rapid habitat loss is

the single most primary cause of

endangerment. Therefore, wildlife can be

victims of human activities resulting in

exposure to novel infections or conditions that

may affect their response to infections they

already harbour (Thompson, 2013).

Diseases caused by parasites are among the

more important factors leading to death of

Pérez Mata et al.

Neotropical Helminthology. Vol. 10, Nº1, ene-jun 2016

and in several states of USA (Rush, 1932;

Sprent, 1950; 1951; Rogers, 1975; Rogers &

Rogers, 1976; Worley et al., 1976).

Baylisascaris transfuga occurs worldwide in

bears (captive or free-ranging bears), and the

species has been reported in wild bears from

USSR (Oshmarin, 1963), Japan (Okoshi et al.,

1962), Indonesia, Syria, and Tibet (Bromlei,

1965), and USA (Addison, 1978). It has also

been reported in captive bears. (Clark et al.,

1969). More recently, Foster et al. (2004)

included the presence of parasites in the

American black bear (Ursus americanus

Pallas, 1780). Parasites have also been

reported in the european brown bear Ursus

arctos Linnaeus, 1758 (De Ambrogi et al.,

2011; Szczepaniak et al., 2012; Visser et al.,

2015), and polar bear Ursus maritimus Phipps,

1774 (Testini et al., 2011). These reported were

from diverse countries including Japan,

Netherlands and Italy. Morphological features

of Baylisascaris transfuga include: cervical

alae present, finelly striated with oral lips

marked by a deep groove, fine dentigerous

ridges present, 66 precloacal papillae and

presence of area rugosa near cloaca (Sprent,

1968; Hartwich, 1989)

The species Baylisascaris shroederi is

considered the most common helminth found

in giant panda and it usually parasitizes the

intestines causing intestinal obstruction,

inflammation, and death (Zhang et al., 2008,

Zhang et al. 2011; Zhang et al., 2012).

According with Hu (2001), most giant pandas

found dead in the wild have been heavily

infected with B. shroederi. Finally, eggs of

supposed Baylisascaris spp. were collected in

faeces of captive spectacled bears from several

US zoos (Schaul, 2006). Although normally

the parasite does not cause major symptoms,

heavy infection could cause illness or even

death (Testini et al., 2011).

Tremarctos ornatus is an endangered species

and little is known about the parasites that can

affect these precious animals. According with

wildlife (Thompson et al., 2010). Among

them, species of ascarids are highly prevalent

and may severely affect the healthy status of

vertebrate hosts. Baylisascaris spp. are

ascarid nematodes affecting different animals.

Sprent (1968), reclassified the ascarid

nematodes from bears of the genus Ascaris and

Toxascaris into Baylisascaris, a genus

reported at that time naturally infecting all

bears species except spectacled bears. Then,

subfamily Ascaridinae (into family

Ascarididae) includes Baylisascaris, Ascaris,

Toxascaris, Parascaris, and Lagochilascaris

(Sprent, 1983; Adamson, 1986).

According with Kazacos (2008), there are

eight recognized species within the genus

Baylisascaris: B. procyonis Stefanski and

Zarnowski, 1951 (syn. Ascaris procyonis

Sprent, 1968), in raccoons; B. columnaris

Leidy, 1856 in skunks; B. melis Gedoelst, 1920

in badgers; B. devosi Sprent, 1952 in martens;

B. schroederi McIntosh, 1939, in the giant

panda (Ailuropoda melanoleuca David, 1869);

B. tasmaniensis Sprent, 1970, in Tasmanian

devils, quolls and native “cats”; B. laevis

Leidy, 1856, in marmots and ground squirrels

and B. transfuga Rudolphi, 1819, affecting

bears. Another two species more recently

described are, B. ailuri for the red panda

(Ailurus fulgens Cuvier, 1825) (Xie et al.,

2011), and B. potosis, in the kinkajou (Potos

flavus Schreber, 1774) (Tokiwa et al., 2014;

Tokiwa et al., 2016).

Baylisascaris spp. can cause severe larva

migrans syndrome in accidental host. By

instance, humans are accidental intermediate

hosts for B. procyonis. Infection follows the

ingestion of B. procyonis eggs containing

infective second-stage larva. In humans, B.

procyonis larvae undergo aggressive tissue

migration that includes the CNS and eyes.

(Gavin et al., 2005)

Baylisascaris has been reported in grizzly and

black bears in Canada (Choquette et al., 1968)

Baylisascaris venezuelensis n. sp. from Tremarctos ornatus

Neotropical Helminthology. Vol. 10, Nº1, ene-jun 2016 Pérez Mata et al.

yeyuno-ileon of varicous aspect. Observation

of the intestine revealed that varicose aspect

was due to a large amount of nematodes which

were filling the intestinal lumen and were

visible under serosa (Fig 1). Stomach was also

filled with these parasites. At macroscopic

examination these nematodes were ascaroid-

like, whitish and large in size (10 to more than

20 cms long). The cause of dead was

established as a severe parasitism on the basis

of bad body condition and the huge amounts of

parasites found. Animal & carcasse was

conserved for Bailadores Municipality for

taxidermal purposes. Only a few specimens of

the nematodes (one female and one male) were

conserved in 70% alcohol and sent by the

Veterinarian to Helminthology Laboratory,

Center for Research in Veterinary

Parasitology, School of Veterinary Sciences,

Universidad Central de Venezuela (Maracay,

state of Aragua, Venezuela, 800 km away) for

p a r a s i t o l o g i c a l a n d m o l e c u l a r

characterization.

Parasitological procedures

Two nematodes, a male and female conserved

in 70% isopropyl alcohol were received at the

Helminthology laboratory. Male length was

10, 2 cm; female length, 25 cm. Parasites were

clarified in lactophenol and identification was

made based on Sprent (1968), CIH Keys

(Chabaud, 1989; Hartwich, 1989), and

Mozgovoi (1953). Parasites were studied

under a stereomicroscope (Karl Zeiss® Stemi

v2000), and trinocular microscope (Nikon®

E200) with a digital camera attached. Pictures

of male specimen were also taken with a digital

image analyzer system (Nikon®) at the

Zoology Museum and Institute, “MIZA”

(Agronomic Engineering school, UCV), fixed

with pins over a paraffin block. A 2 cm

fragment from the middle part of the body of

the male specimen was placed in ethanol 99%

for molecular techniques No sectioning of the

parasite was made for morphological

description because only two specimens were

available. Thus, morphological examination

data taken from captive animals, ascarids are

the most common parasites affecting T.

ornatus (Wolff, 1988; WildPro, 2013;

Figueroa & Stucci, 2003). Figueroa (2015)

found ascarids ova in 28 fecal samples from

free ranging T. ornatus in Laquipampa

Wildlife Refuge (Peru). The aim of this study

was to describe, for the first time, a fatal case of

natural infection by Baylisascaris sp. in a free-

ranging young T. ornatus from a natural

reserve in the Andean Mountains of Venezuela

and to describe the morphological and

molecular features of this that we consider a

new species of Baylisascaris, whose control

can contribute to the conservation of the

spectacled bear in South America.

A little female specimen of a spectacled bear

(T. ornatus) was found dead by National Guard

Officers during a patrol in the National Park

India Carú, Bailadores town, Rivas Dávila

Municip ality, M érida, Venezuela

(Coordinates: 8.2562189—71.8254101, mean

altitude 1830 meters over sea level). The

Mountain region of this park belongs to South

American Andes.

Since this is an endangered species protected

by law, a local veterinarian was called to

perform necropsy of the bear and to certify (if

possible) the cause of dead. Necropsy was

performed at the Experimental Laboratory of

the “Universidad Politécnica Kleber Ramírez,

Campus Bailadores”. The specimen was a little

female bear, 89 cm length, 40 cm head

diameter, 26 kg of weight, 59 cm height.

External exploration did not reveal fracture,

trauma, gunshot wounds, blood, or any

secretion through natural openings. Internal

examination at macroscopic level included:

Thoracic cavity: lungs with congestion and

hemorrhagic focus. Abdominal cavity: Small

bowel enlarged, with several segments of

MATERIAL AND METHODS

sequences are as partial sequences. Therefore,

genealogy and genetic relatedness of the

Baylisascaris sp. from the Venezuelan

spectacled bear was accomplished based on

three independent alignments constructed

using sequences herein determined and

sequences retrieve from the GenBank:

Alignment 1: ITS1 rDNA sequences; this

alignment was constructed using complete

ITS1 rDNA sequences from the new specimen

from spectacled bear from Venezuela (three

cloned sequences) plus diverse sequences

from the main related roundworm parasites

retrieved from the GenBank. ITS1 sequence

from Bunostomum phlebotomum Railliet,

1900 was used as outgroup. The aim of this

alignment was to positioning the new

sequences from the roundworm from

Venezuelan bear in a genealogy including

parasites from the genus Baylisascaris,

Ascaris, Parascaris, Raphidascaris,

Krefftascaris, Toxocara, Hysterothylacium,

C o n t r a c a e c u m , A n i s a k i s a n d

Pseudoterranova. Accession numbers of

sequences retrieved from the GenBank are

showed in Figure 2A. Alignment 2: ITS1

rDNA sequences; this alignment was

constructed with ITS1 rDNA sequences from

exclusively species of the Baylisascaris genus.

The aim was to assess the relatedness of the

new sequences determined in this study with

others species of the Baylisascaris genus.

Alignment 3: ITS2 rDNA sequences; this

alignment was also constructed exclusively

with sequences (partial or complete ITS2

rDNA) from species of the Baylisascaris

genus and the aim was to assess the relatedness

of species within the Baylisascaris genus and

to compare with ITS1 analysis. Accession

numbers of ITS1 and ITS2 rDNA sequences

retrieved from the GenBank are showed in

Figure 3A and 3B. Ascaris summ ITS1 or ITS2

rDNA sequences retrieved from the GenBank

was used as outgroup for alignment 2 and 3,

respectively. Finally, an alignment displaying

the sequence polymorphism within the

conserved region 5.8S rDNA of Baylisascaris

was limited to those measures which did not

imply that procedure (SanMartín et al., 1992;

Esquivel, 2011).

A complete specimen (female) and two

fragments (caudal and cervical ends) of a male

were conserved and deposited on

Helminthological collection of the Center for

Research in Veterinary Parasitology “Dr.

Manuel Antonio Rivera Acevedo” (CIPV-

MARA), School of Veterinary Sciences,

Universidad Central de Venezuela. (Catalogue

number: CIPVMARA2010-N-EX-999).

Molecular procedures

Total DNA was extracted from a male parasite

by incubation of a small segment of the middle

part of the specimen (50 mg) in a lysis buffer

(1% SDS, 100 mM EDTA pH 8.0, 20 mM Tris-

HCl, pH 8.0 and 350 mg/ml of proteinase K) at

37 ºC for 18 h. Then, sample was centrifuged at

14000 g for 5 min. and DNA was purified using

Wizard DNA Purification Systems

(Promega ). An aliquot of 1-2 µL was used as

template for amplification of the whole ITS

rDNA (wITS: ITS1-5.8S-ITS2) using primers

and reaction conditions previously described

(Gasser et al., 1996). PCR products from three

independent reactions were pooled, resolved

by agarose gel (1.5%) electrophoresis, stained

with ethidium bromide (0.5 µg/ml), excised

from the agarose gel, purified with a Spin-X

® ®

kit (Costar ), and cloned with a TA-Cloning

kit (Invitrogen ). The sequences of 5 clones

were determined by automated sequencing and

clones representing the polymorphism

detected were selected for genetic analyses.

Sequences were aligned using the ClustalX

program (Thompson et al., 1997) and refined

manually.

The availability of wITS rDNA sequences

from Baylisascaris species at the GenBank is

limited and both for this genus and other

related ascarid parasites (roundworm)

majority of complete ITS rDNA sequences are

restricted to ITS1 rDNA; whereas ITS2 rDNA

Neotropical Helminthology. Vol. 10, Nº1, ene-jun 2016 Baylisascaris venezuelensis n. sp. from Tremarctos ornatus

Neotropical Helminthology. Vol. 10, Nº1, ene-jun 2016 Pérez Mata et al.

PAUP* v. 4.0b10 (Swofford, 2002) with 100

replicates of random addition sequence,

followed by branch swapping (RAS–TBR), as

previously described (Li et al., 2012; Zhao et

al., 2012; Tokiwa et al., 2015). Distance

matrices were produced using the uncorrected

p distance. The alignments of the ITS rDNA

sequences are available from the authors by

request.

Morphological identification

Nematodes were identified as Order

Ascaridida, superfamily Ascaridoidea based

on size (male: 10,5 cm; female: 25 cm) and

presence of three characteristic lips at the

cephalic end, genus Baylisascaris sp., first

time reported naturally infecting T. ornatus in

Venezuela, on the basis of:

1. host

2. denticular ridges on internal surface of lips

and a marked groove surrounding lips, with

cervical alae conspicuous, slightly curled and

striated (Fig. 4, 5)

3. Caudal extremity of male showing equal

stout, not too long spicules (0,9 mm) and

presence of area rugosa near cloaca of male

(Fig. 6)

4. Posterior margin rounded, Number of

papillae (Fig. 7): as in several species of

Baylisascaris, there are a high number of pre-

cloacal papillae. For our specimen we found 44

pre-cloacal papillae, differing from B.

transfuga, the specific species of bears, which

has 66 (according with Sprent, 1968). Table 1

and Table 2 summarizes the major features of

our specimens, being the remarkable

difference in the number of precloacal

papillae.

Molecular analysis

In this study we evaluated polymorphic

sequences from the internal transcribed spacer

species was constructed. The aim was to reflect

the single nucleotide polymorphisms (SNPs)

in this highly conserved rDNA region that

support the new spectacled bear specimen as a

new Baylisascaris species (Fig. 2B).

DNA sequence analyses

Entire cloned sequences (ITS1+5.8S+ITS2)

were initially used as query for sequence

identity using the BLAST program (nucleotide

bl a st) a t t h e N C BI Ho m e P age

(http://www.ncbi.nlm.nih.gov/) using the blast

algorithm with the standard searching setting.

Newly obtained sequences were aligned as

described above and used for phylogenetic

analyses as following:

Alignment 1 was used for construction of the

Network genealogy using the Neighbor-Net

method with Kimura 2 parameters

implemented in Splits Tree4 V4.10 (Huson &

Bryant, 2006). Internode support was

estimated by performing 100 bootstrap

replicates using the same parameters

optimized for network inferences.

Furthermore, this alignment was used for

genealogy reconstruction using Bayesian

method. Alignments 2 and 3 were used for

genetic relatedness among ITS1 or ITS2 rDNA

sequences from Baylisascaris species. These

sequences were assessed by Bayesian, ML and

Maximum Parsimony (MP) methods.

Bayesian analysis was done using MrBayes

v3.1.2 (Ronquist & Huelsenbeck, 2003). Tree

searches employed GTR plus gamma and

proportion of invariable sites. The first 25% of

the trees from 100000 generations were

discarded as burn in. ML trees were inferred

using RAxML v.7.0.4 (Stamatakis, 2006)

using the GTRGAMMA model, gamma shape

parameter and proportion of invariable sites,

with maximum parsimony starting trees.

Model parameters were estimated in RAxML

over the duration of the tree search. Nodal

supports were estimated using 500 replicates

using a rapid bootstrapping algorithm. MP and

bootstrap analyses were performed using

RESULTS

clones) and 435 (one clone). These lengths

were different from the shorter sequences of B.

transfuga and B. schroederi (428 bp).

Similarly, the new ITS2 rDNA sequences

determined (two polymorphic sequences out

of the five clones) varied from 336 to 338 bp

length, differing from sequences from B.

transfuga (HM594951) and B. schroederi

(JN210911, JN210912, EU642816), which

were 325 bp length. Variations in the length of

poly-A motifs in both ITS1 and ITS2 rDNA

and in the number of microsatellite repeats in

the ITS2 region were the source for length

variations of the new sequences from the

Venezuelan bear roundworm when compare to

B. transfuga and B. schroederi. Similarly, an

insertion of a simple sequence motif (ATA) in

the ITS1 rDNA influenced the length

variations. However, several SNPs distributed

in both the ITS1 and ITS2 rDNA split B.

transfuga and B. schroederi from the

Baylisascaris specimen from the spectacled

bear from Venezuela.

The more conserved 5.8S rDNA region from

the Venezuela specimen was 155 bp length.

This length was identical for all Baylisascaris

species available at the GenBank, including B.

transfuga, B. schroederi, B. procyonis and B.

columnaris (Figure 2B). The 5.8S sequence

from B. potosis available at the GenBank is a

partial sequence precluding an accurate

comparison. Two SPNs located in the highly

conserved 5.8S region separated the new

sequences from all other Baylisascaris

species, i.e: transitions at positions 27 (T by C)

and 104 (A by G) (Fig. 3B).

Analysis of sequence polymorphisms within

the wITS rDNA

For analysis of sequence polymorphisms

(wITS rDNA) the ambiguously aligned

regions were excluded by mean of Gblocks

(http://molevol.cmima.csic.es/castresana/Gbl

ocks_server.html). The genetic analysis

disclosed significant divergences among

Baylisascaris sp. from T. ornatus from

(ITS1 and ITS2 rDNA), as well as sequences

from the highly conserved 5.8S gene.

Molecular identification and wITS sequence

analysis

In this study were sequenced five clones of the

entire ITS rDNA (wITS), which spanning the

regions of the ITS1+5.8S+ITS2. A BLAST

search of wITS sequences here determined

showed 90% and 89% sequence identity with

B. transfuga from T. maritimus (Italy) and B.

schroederi from Ailuropoda melanoleuca

(China), respectively (accession numbers

HM594951 and JN210911-JN210912). This

initial BLAST search allowed us to suggest

that the spectacled bear parasite belong to the

Baylisascaris genus. However, the percentage

of identity of the new sequences with others

species of the Baylisascaris, as well as the host

and geographical origin of the new specimen

suggests a putative new genotype or species

within this genus. Therefore, the new

sequences were used for genealogy

reconstruction and relatedness analysis

The wITS rDNA from the spectacled bear from

Venezuela was 927-930 base pair (bp) length, a

total of 17-20 bp longer than those from B.

transfuga (HM594951) and B. schroederi

(JN210911, JN210912), which displayed

sequences of 910 bp. Except for the isolates

from B. transfuga and B. schroederi referred

above, no other Baylisascaris species has

complete ITS1 or ITS2 rDNA sequences for

length and sequences comparison at the

GenBank.

Three out of five wITS rDNA clones

determined from the spectacled bear specimen

were polymorphic. However, the

polymorphism detected was limited to both

length variations in a poly-A region within the

ITS1 rDNA and an additional microsatellite

repeat (GA) within the ITS2 rDNA. Thus, the

divergence among cloned sequences was

~0,1%. ITS1 rDNA sequences spanning

nucleotides 1 to 432 (two clones), 434 (two

Neotropical Helminthology. Vol. 10, Nº1, ene-jun 2016 Baylisascaris venezuelensis n. sp. from Tremarctos ornatus

Neotropical Helminthology. Vol. 10, Nº1, ene-jun 2016 Pérez Mata et al.

transfuga, B. procyonis, B. columnaris and B.

schroederi). The 5.8S from B. potosis

(accession number KF680774) was not used

for comparison because the first 53 nucleotids

are missing. Interesting, three SNPs at the

highly conserved 5.8S separate the spectacled

bear parasite from B. transfuga (from U.

maritimus / U. arctos) and B. schroederi (from

Ailuropoda melanoleuca), which share the

same 5.8S sequences (transitions at positions

27, 104, 114). Similarly, three SNPs split the

spectacled bear parasite from B. columnaris

(from Mephitis mephitis Schreber, 1776) and

B. procyonis (from Procyon lotor Linnaeus,

1758), which also share 5.8S sequences

(transitions at positions 27, 104, 105). At least

one SNP separate B. potosis from the new

Venezuelan roundworm specimen even when

the 5.8S sequence from B. potosis is

incomplete (Fig. 2B).

Venezuela and sequences from other

Baylisascaris species. The divergence among

the five wITS clones of Baylisascaris sp.

herein determined was small (~0.1% internal

divergence). However, high divergences

separated these sequences from those from B.

transfuga (8.2%), B. schroederi (9.4%) and, B.

procyonis (10%). The divergence among B.

transfuga and B. schroederi was 2.8%; while

larger divergences separated B. procyonis from

B. schroederi (~8%) and B. transfuga (~8%).

Sequences from the wITS (ITS1+5.8S+ITS2)

from B. columnaris and B. potosis available at

the GenBank were too incomplete for

comparative purposes.

Sequence polymorphisms regarding to the

5.8S regions revealed ~2% of divergence

among Baylisascaris sp. from spectacled bear

and all the other 5.8S sequences analyzed (B.

Figure 1. Intestine with varicose aspect due to a large amount of nematodes (Baylisascaris venezuelensis) filling the intestinal

lumen and visible under serosa.

Figure 2. (A). Network genealogy of the ITS-1 rDNA sequences of parasites in the Order Ascaridida showing the position of

Baylisascaris venezuelensis n. sp., inferred using the Neighbour-Net method with the K2P parameter and Bayesian analysis.

Bunostomum phlebotomum (GenBank GQ859497) was used as outgroup. Numbers at nodes represent: Posterior probabilities for

the Bayesian analysis (first value) and bootstrap values from 100 replicates for the Network analysis (second value). (B).

Alignment of the 5.8S rRNA gene sequences from Baylisascaris venezuelensis n. sp and comparison with homologues from other

Baylisascaris species. This figure shows the single nucleotide polymorphisms (SNPs) that split the new Baylisascaris species

from Tremarctos ornatus from Venezuela from the other related Baylisascaris spp. Dots represent identical nucleotides, while

“N” represent not determined ones.

Neotropical Helminthology. Vol. 10, Nº1, ene-jun 2016 Baylisascaris venezuelensis n. sp. from Tremarctos ornatus

Figure 3. Phylogenetic relationships of Baylisascaris species inferred by Bayesian (B), Parsimony (P) and Maximum likelihood

(ML) analysis based on ITS-2 rDNA (A) and ITS-1 rDNA (B) sequences, with Ascaris summ (GenBank AB571302) as outgroup.

Numbers at nodes represent: Posterior probabilities for the Bayesian analysis (first value), maximum parsimony (second value)

and maximum likelihood analyses (third value).

Neotropical Helminthology. Vol. 10, Nº1, ene-jun 2016 Pérez Mata et al.

Figure 4. Baylisascaris sp. from T. ornatus (Venezuela). Head (lateral view). Female.

Figure 5. Baylisascaris sp. form T. ornatus (Venezuela). Head. Detail of groove surrounding lips and dentigerous ridges.

Neotropical Helminthology. Vol. 10, Nº1, ene-jun 2016 Baylisascaris venezuelensis n. sp. from Tremarctos ornatus

Figure 7. Baylisascaris sp. from T. ornatus (Venezuela). Male tail showing pre-cloacal papillae

Figure 6. Baylisascaris sp. from T. ornatus (Venezuela). Male tail showing round end (with a little process) and two short, stout

spicules.

Neotropical Helminthology. Vol. 10, Nº1, ene-jun 2016 Pérez Mata et al.

Table 1. Some morphological features of Baylisascaris sp. infecting T. ornatus from Bailadores (Venezuela)

compared with six different species of Baylisascaris*.

Species Baylisascaris sp.

(Venezuela) B.

transfuga*

melis*

devosi*

columnaris*

procyonis*

schroederi*

Male length (cm) 10,2 10,2

12,7

12,3

9,0

9,8

Female lenght (cm) 25 24,0

22,4

28,5

22,5

20,0

12,5

Alae Salient, striated Salient

Salient

Vestigial

Dentícules

Equilateral

triangle Equilateral

triangle

Equilateral

triangle

High

triangles

Equilateral

triangle

Equilateral

triangle

Inconspicuous

Shape of median lobe of

lips Saddle saddle

Saddle

% leght anterior to

vulva 40 37

Female tail

(mm) 2

Button (round) 1,4

Button

1,6

Button

0,89

Button

0,94

Button

1,4

Point

Spicules

(mm) 0,9 0,93

9,9

Male tail

(mm) 0,5 0,6

0,52

Pre

cloacal papillae (nº) 44 66

Pre

cloacal area Posterior margin

round (with a little

process)

Posterior

margin

round

Posterior

margin

round

Posterior

margin

round

Pointed

round

*: measures recorded from Sprent (1968).

Table 2. Additional measures recorded from Baylisascaris sp. infecting T. ornatus from Bailadores (Venezuela).

Features

Lenght

(mm) Width

(esophageal

región, mm)

Ratio

Width /

Lenght

Width at

anal/cloacal

región (mm)

Lenght

of tail Ratio

Lenght of

tail/Lengh

Ratio Lenght of

tail/width at anal

region

Male 102 3mm 0,029 0,8 0,5 0,005 0,625

Female 250 4,1mm 0,016 2,1 2 0,008 0,952

DISCUSSION

The nematode genus Baylisascaris (order

Ascaridida, superfamily Ascaridoidea)

contains ten relatively host-specific, parasite

species of carnivores, omnivores, herbivores,

carnivorous marsupials or rodents (Rogers &

Rogers, 2013; Tokiwa et al., 2014).

Baylisascaris transfuga is considered the

specific ascarid of bears, and has been

identified naturally infecting all species of

bears, excepting spectacled bear (Choquette, et

al., 1968; Sprent, 1968; Crum et al., 1978).

Reports of the presence of Baylisascaris

transfuga or undetermined ascarids infecting

the spectacled bear was made on the basis of

fecal examination of captive individuals

(Schaul, 2006, Figueroa, 2015) and Acosta et

al. (2015) did no found evidence of helminth

infection in captive spectacled bears from Zoo

“Las Leyendas” (Peru). We had several

limitations for the morphological study, i.e. we

only had two specimens, and because of these a

careful examination of internal structures was

not possible, since we need the specimens as

holotype and allotype. Even though, the

morphological features of Baylisascaris

Neotropical Helminthology. Vol. 10, Nº1, ene-jun 2016 Baylisascaris venezuelensis n. sp. from Tremarctos ornatus

Neotropical Helminthology. Vol. 10, Nº1, ene-jun 2016 Pérez Mata et al.

from Venezuela within the genus

Baylisascaris, near to ascarid of the genus

Ascaris and Parascaris, which were the sister

groups. The network genealogy using ITS1

rDNA sequences from B. transfuga, B.

procyonis, B. schroederi and B. columnaris

from the GenBank, as well as diverse

sequences from related parasites grouped the

spectacled bear roundworm sequences

together with all Baylisascaris sequences

analyzed in a monophyletic assemble, the

genus Baylisascaris. Regardless of the

analytical approach (Bayesian, ML and NJ), all

methods yielded similar topologies and the

same relationships for all major clades which

were highly supported (Fig. 2A). According to

network topology, the spectacled bear

specimen clustered closer to B. transfuga and

B. schroederi; while B. procyonis and B.

columnaris conformed a separated subcluster

within the genus Baylisascaris. These intra

genus relatedness were confirmed by

independent ITS1 (Figure 3B) and ITS2

(Figure 3A) rDNA analysis. In this later, partial

sequences clustered B. potosis near to B.

procyonis and B. columnaris; whereas

sequences from B. ailuri, B. transfuga and B.

schroederi were positioned as independent

species nearest to sequences from

Baylisascaris from the Venezuelan bear. Tree

topologies were identical regardless the

analytical approach (Bayesian, ML and

Parsimony) and clades were highly supported.

Interesting, the relationships of Baylisascaris

species using ITS1 and ITS2 rDNA sequences

displayed a closed association of the new

Baylisascaris species from the spectacled bear

Tremarctos ornatus with B. transfuga from the

brown bear (Ursus arctos) and the polar bear

(Ursus maritimus), B. schroederi from the

giant panda (Ailuropoda melanoleuca) and B.

ailuri from the red panda (Ailurus fulgens).

These results display an interesting

phylogenetic conservatism in the used of

mammals host by this group of Baylisascaris

species.

Nowadays, beside traditional features from

species are particularly externals, and remain

in very small details, like number of precloacal

papillae, shape of denticles, or shape of tail in

posterior margin (species described by Sprent,

1968), or even more difficult, can be

distinguished like in B. potosis just by the

position of the male phasmidial pole (Tokiwa

et al., 2014). Morphological identification of

parasites from wildlife can be a difficult, time-

consuming task and usually needs expertise

and the aid of DNA analysis (Sepúlveda &

Kinsella, 2013). Regarding Baylisascaris

genus, identification of most recently

described species and confirmation of the

older ones are highly supported by molecular

studies (Zhang et al., 2008; Testini et al., 2011;

Zhang et al., 2011; Zhang et al., 2012; Zhao et

al., 2012; Zhou et al., 2013; Tokiwa et al.,

2014; Tokiwa et al., 2015; Visser et al., 2015).

Analyses based on molecular characters rather

than morphological ones are needed in the

study of these ascarid nematodes phylogeny to

evaluate the previous findings. The application

of molecular approaches may provide

important evidence needed to clarify the

taxonomic status and to determine the

relationships among these ascarid nematodes

(He et al., 2013). Although our nematodes

primarily appeared to belong to the species B.

transfuga, a careful examination of the caudal

end of male revealed differences with features

described for B. transfuga. i.e: number of pre-

anal papillae. According with Sprent (1968) T.

transfuga has 66 pre-cloacal papillae while our

specimen has 44 papillae. Until the present

work no evidence of natural infection with

Baylisascaris sp. in wild specimens of T.

ornatus had been reported. Our molecular

studies supported the importance of the

morphological differences we found between

our specimens and B. transfuga.

Genealogy and genetic relatedness using ITS

rDNA sequences

In this study, a genealogy network analysis

considering several related nematode parasites

unambiguously positioned the new sequences

naturally occurring of other Baylisacaris sp. in

humans, all Baylisascaris species are

potentially zoonotic. Concerns also raised for

the more unknown potentially zoonotic

Baylisascaris species, such as B. transfuga

(Visser et al., 2015) Thus, the zoonotic

potential of Baylisascaris, and the existence of

human invasion of the habitat of this

endangered animal (even in National Park

areas), in the venezuelan Andes represents a

potential human health risk which must be

taken in account.

Proposed new species:

Baylisascaris venezuelensis Pérez, García &

Gauta, 2015

Host type: Tremarctos ornatus

Location: Bailadores (Mérida, Venezuela)

Voucher specimens: deposited on the

Helminthological collection of the Center for

Research in Veterinary Parasitology “Dr.

Manuel Antonio Rivera Acevedo” (CIPV-

MARA), School of Veterinary Sciences,

Universidad Central de Venezuela, under the

Catalogue number: CIPVMARA2010-N-EX-

999.

Morphological features: Nematoda,

Ascaridida.

Holotype: Male: stout nematodes, 10.2 cm

length, posterior end slightly curled dorsally,

tail round finished, with a little finger-like

process; spicules: 0.9 mm, area rugosa

present, 44 pre-cloacal papillae; anterior end: 3

lobes typical ascaroid-like with marked groves

between lips and presence of dentigerous

ridge, (triangle), cervical alae conspicuous,

slightly curled and striated. Specimen divided

into two fragments (anterior and posterior,

lacking a two cm fragment in the middle half of

the body (used in molecular analysis).

Allotype: Female: big, stout nematodes,

whitish, pale yellow, 25 cm long. Head: 3 lobes

with markedly surrounded dentigerous ridges,

cervical alae conspicuous, slightly striated and

curled.

morphology, host and geographic origin,

disease epidemiology and disease patterns and

others, the description of new species or

genotypes of parasites should considered

molecular data of conserved and/or

polymorphic DNA sequences from nuclear or

mitochondrial origin.

Morphological characterization of our

nematode showed that it belongs to

Baylisascaris genus, worms first time

recovered from wild spectacled bear, and

clearly differing from T. transfuga (typical

ascarid from bears) according with the number

of pre-cloacal papillae. Molecular study of our

nematodes from T. ornatus evaluated

polymorphic sequences from the internal

transcribed spacer (ITS1 and ITS2 rDNA), as

well as sequences from the highly conserved

5.8S gene. Venezuelan specimen was clearly

separated from other species of Baylisascaris

in each of the analysis performed. Considering

the endemicity of the host species, its restricted

geographical location, with isolate populations

exhibiting different genotypes depending on

the geographical location (Ruíz-Martínez,

2003; Ruíz-Martínez et al., 2005), the origin of

parasites (natural infection from a wild animal)

and the morphological divergences at the

caudal extreme of male, besides the

remarkable results of the molecular genetic

analysis and phylogenetic placement (with a

new genotype clearly different from other B.

transfuga, genotypes recorded), all together

strongly suggest that this is a new

Baylisascaris species for the South American

spectacled bear T. ornatus, and we suggest the

name of Baylisascaris venezuelensis

considering this is the country in which the

parasite was found. Beyond the parasitological

finding, it becomes a new concern for

venezuelan species-level conservation efforts

for spectacled bear. Furthermore, it is known

that Baylisascaris procyonis is the only well

documented and most frequently cause of

human and animal baylisascariosis. Even

though there is no unequivocal evidence of

Neotropical Helminthology. Vol. 10, Nº1, ene-jun 2016 Baylisascaris venezuelensis n. sp. from Tremarctos ornatus

100

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