Acta Tropica 137 (2014) 95–98

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Acta Tropica

jo urnal homepage: www.elsevier.com/locate/actatropica

Detection of Paragonimus mexicanus () metacercariae in

from Oaxaca, Mexico

a a a

Jaime Vargas-Arzola , Aristeo Segura-Salvador , Leobardo Reyes-Velasco ,

b c c

Dylan L. Díaz-Chiguer , Adrián Márquez-Navarro , Gloria León-Avila ,

d c c

Gabriela Ibanez-Cervantes˜ , Alejandro D. Camacho , Rosa Ma. Sánchez-Manzano , c,∗

Benjamín Nogueda-Torres

a

Facultad de Ciencias Químicas, Universidad Autónoma “Benito Juárez” de Oaxaca, Oaxaca 68120, Mexico

b

Instituto de Seguridad y Servicios Sociales para los Trabajadores del Estado, ISSSTE, México DF 06720, Mexico

c

Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, México DF 11340, Mexico

d

Centro Nacional de la Transfusión Sanguínea, México DF 07360, Mexico

a r t i c l e i n f o a b s t r a c t

Article history: Metacercariae of Paragonimus mexicanus were collected in crabs Tehuana guerreroensis (Rathbun, 1933)

Received 4 December 2013

in the municipality of Putla, Oaxaca, Mexico. Metacercariae were found in 20.8% of the crabs collected,

Received in revised form 3 May 2014

with an average of 1.9 metacercarie per . Stained metacercariae showed the specific characteristics

Accepted 9 May 2014

of P. mexicanus by morphology and sequencing a fragment of the 28S ribosomal gene obtained by PCR.

Available online 16 May 2014

These findings reveal that T. guerreroensis is an intermediate host for P. mexicanus; this new report is

relevant considering the potential risk of transmission in the states of Oaxaca and Guerrero, Mexico.

Keywords:

© 2014 Elsevier B.V. All rights reserved.

Paragonimus mexicanus Metacercariae Crabs

Tehuana

1. Introduction Perú, Ecuador, Costa Rica, Panamá and Guatemala. In México,

this parasite has been found in wild from Colima, Chia-

Paragonimiasis is a food-borne parasitic disease of humans, pas, Hidalgo, Michoacán, Nayarit, Puebla, San Luis Potosi, Tabasco,

other , molluscs and ; it is caused by flukes of Yucatán, Veracruz and State of México (Lamothe-Argumedo, 1985).

the genus Paragonimus spp. Human is distributed Several species of the family and

across three continents: South and Central America, East Asia and one of the family Trichodactylidae serve as second intermediate

West Africa. Typical symptoms of human paragonimiasis include hosts (Blair et al., 1999).

fever, cough, eosinophilia and hemoptysis, which together can be At least 13 genera and 48 species of the family Pheudothel-

misdiagnosed as tuberculosis. Praziquantel is the drug of choice for phusidae have been described in México, as summarized elsewhere

paragonimiasis; however, other drugs have been used (Keiser and (Rodríguez and Magalhães, 2005). P. mexicanus metacercariae

Utzinger, 2010). Paragonimus spp. use snails as first intermediate have been found in crustaceans such as Pseudothelphusa dilatata

hosts and decapod crustaceans (crabs and freshwater shrimp) as (Lamothe-Argumedo et al., 1977), Ptychophallus tristani and P.

second intermediate hosts. costaricensis (Brenes et al., 1980; Mongue et al., 1985), Odontothel-

Infection occurs when man ingests either raw or uncooked phusa maxillipes and Raddaus tuberculatus (Lamothe-Argumedo,

crustaceans infected with metacercariae or meat from paratenic 1984), Pseudothelphusa americana belliana, P. nayaritae and P. ter-

hosts. More than 30 species of the genus Paragonimus have been restris (Lamothe-Argumedo, 1995), all of which serve as second

described; of these, 10 species have been reported to infect intermediate hosts. In many regions of Mexico, as in the state of

humans. Paragonimus mexicanus is located in México, Colombia, Oaxaca, people traditionally include wild crabs and other crus-

taceans as part of their diet. Improper cooking of these crabs

represents a potential risk for infection due to ingestion of Parag-

∗ onimus spp. metacercariae. The aim of this study is to isolate and

Corresponding author. Tel.: +52 5557296000x62399.

evaluate the presence of P. mexicanus from crabs collected in the

E-mail addresses: [email protected], [email protected]

(B. Nogueda-Torres). state of Oaxaca, Mexico.

http://dx.doi.org/10.1016/j.actatropica.2014.05.004

0001-706X/© 2014 Elsevier B.V. All rights reserved.

96 J. Vargas-Arzola et al. / Acta Tropica 137 (2014) 95–98

Fig. 1. Map of Mexico country and localization of Putla Villa de Guerrero, state of Oaxaca, Mexico.

2. Materials and methods 2.3. DNA extractions from metacercariae

2.1. Source of crabs DNA was extracted from approximately 5 mg of metacercariae.

DNA was purified using the DNeasy Blood & Tissue Kit (Qiagen,

Crabs were collected at San Miguel Reyes, municipality of Putla Valencia, CA, USA) and its concentration was measured using a

◦  

Villa de Guerrero, State of Oaxaca, Mexico (97 55 45 West and, nanoDrop 2000 (Thermo Scientific Waltham MA, USA). DNA from

◦  

17 01 33 North at an altitude of 783 m.a.s.l.) (Fig. 1). A total of 120 uninfected crabs maintained and bred in the laboratory was used

◦

crabs (Fig. 2) of various sizes were collected between February 16th as negative control. All DNA samples were stored at −20 C until

and March 13th 2011, in small freshwater streams. Water tempera- use.

◦

ture at the time of capture was 21 C; pH was 7.0. Crabs were placed

in plastic containers and transported alive to the laboratory.

2.4. PCR assay

2.2. Isolation of metacercariae

The DNA extracted was amplified in a 25-␮l reaction well

using 100 ng of DNA template, 0.8 ␮M each of forward and reverse

In the laboratory, crabs were allowed to acclimatize for two days

primers (final concentration), and Master Mix (Roche). Amplifi-

and then weighed and measured with a caliper. Afterwards, crabs ®

◦ cation was run in a Tc-3000 (TECHNE) . Cycling was performed

were killed by thermal shock (2 C for 10 min) fixed in 5% formalin ◦

as follows: initial DNA denaturing (94 C, 5 min), followed by 30

and then placed in plastic containers. Crabs were then dissected: ◦ ◦

cycles each of denaturing (92 C, 30 s), annealing (61 C, 30 seg),

they were cut in half from the cephalothorax in longitudinal sec- ◦ ◦

extension (72 C, 1 min). The final extension was 72 C for 4 min.

tion in order to extract the gills, digestive system and ontocele.  

The primers 28S-F 5 -GAGGGTGAAAGGCCCGTGGG-3 and 28S-R

This material was placed in Petri dishes with saline, the tissue was  

5 -ACGCATGCACACACCTCRAGCCG-3 were designed in a conserva-

finelly sliced with scalpel and carefully examined under a stereo

tive region bracketing a variable region of approximately 630 bp of

microscope in search of Paragonimus spp. When found, metacer-

the 28 S rRNA. Amplicons obtained were analyzed in 1.5% agarose

cariae were rinsed in saline and preserved in 96% ethanol until used.

gel stained with ethidium under UV light. Positive and negative

Individual metacercaria were observed under the microscope.

controls were always included.

2.5. Sequencing and BLAST

The PCR product was sequenced in both strands in the Unit

of Proteogenomics, UNAM, Juriquilla, Mexico. The sequences were

viewed in the Chromas program Lite and refined manually. The final

sequence was analyzed by BLAST in the NCBI server.

2.6. Identification of crabs

Adult crabs were sacrificed by thermal shock, fixed in 5% forma-

lin for 5 days and then preserved in glass jars with 70% ethanol.

Specimens were sexed and identified at the family and genus

levels based on external morphological features using a Nikon

SMZ1000 stereo microscope. Males were dissected and the gono-

pod was extracted to allow the specific identification by taxonomic

keys (Rodríguez and Smalley, 1696; Rodríguez, 1982). The updated

species name was taken from criteria of Alvarez and Villalobos

(1994).

Fig. 2. Image of crab Tehuana guerreroensis collected in Oaxaca, Mexico.

J. Vargas-Arzola et al. / Acta Tropica 137 (2014) 95–98 97

Table 1

Prevalence of Paragonimus metacercariae in crabs Tehuana guerreroensis in a com-

munity of Putla Villa de Guerrero, state of Oaxaca, Mexico.

No. of crabs Average of No. of metacercariae/crab metacercariae detected

Examined Infected

120 25 (20.8%) 1.9 48

3. Results

Of a total of 120 crabs examined, 25 specimens (20.83%) were

positive for Paragonimus spp. metacercariae (Table 1). A total of 48

metacercariae were found, averaging 1.9 metacercariae per host

(range: 1–6 metacercariae per host). Metacercariae were mainly

located in the digestive system, ontocele and gills.

Individual metacercariae were stained and the internal struc-

tures were observed. As seen in Fig. 3, the oral ,

excretory system, acetabulum and blind gut showed typical

morphological characteristics of the Paragonimus genus. The

metacercariae were oval (890 ± 195 × 418 ± 94 ␮m) covered with

a spinosed tegument; oral sucker is subapical (93 ± 17 ␮m) and

central acetabulum (diameter 66 ± 12 ␮m); pharynx rectangu-

lar (110 ± 12.3 × 123 ± 12.3 ␮m, base) and intestinal ceca ending

blindly and extended to the end of the body.

The identification of P. mexicanus was confirmed by sequencing

a fragment of the 28S ribosomal gene (Fig. 4).

In Figs. 5 and 6 we show the caparace and left gonopod that allow

the identification of crabs. We determined that crabs belong to

Tehuana guerreroensis (Rathbun, 1933) (Crustacea, Brachyura, Pseu-

dothelphusidae) (= Pseudothelphusa guerreroensis Rathbun, 1933).

Fig. 3. A metacercaria of Paragonimus mexicanus found in the crab Tehuana guer-

reroensis from Putla Villa de Guerrero, state of Oaxaca, Mexico. (1) Oral sucker, (2)

excretory system, (3) acetabulum, (4) blind gut. Scale bar represent 100 ␮m.

4. Discussion

Foodborne trematode infections are still an emerging pub- the diet have led humans to explore and in many cases alter the

lic health issue. These are closely related to the proximity of micro- and macro-environmental conditions resulting in the emer-

human settlements to freshwater bodies. Worldwide, an estimated gence or re-emergence of parasitic (Macpherson, 2005).

750 million people are at risk of infections with foodborne trema- The implementation of common parasitological techniques sup-

todes, and at least 292.8 million humans are specifically at risk of ported by molecular methods such as PCR and DNA sequencing may

infection with the lung fluke Paragonimus spp. (Keiser and Utzinger, be useful in the identification of parasites from different sources,

2005, 2009). This parasite can cause zoonosis in humans when including local fauna, involved in the transmission of parasites.

meat infested with it is consumed raw or undercooked. Although Identification of P. mexicanus infecting crabs T. guerreroensis

these parasites occur in Southeast Asia and Western Pacific regions from the community of San Miguel Reyes, Municipality of Putla

due to aquaculture and traditional cooking practices, some of them Villa de Guerrero, in the State of Oaxaca, Mexico, lead us to con-

may emerge in other continents through international trade and sider this crab species as a second intermediate host. The site of

improved transportation and distribution systems (Dorny et al., Putla is close to the border with the state of Guerrero. Rodríguez and

2009). It has been proposed that “the rising demands on natural Smalley (1696) and Alvarez and Villalobos (1994), report the locali-

resources increase the likelihood of encountering environments ties of Copanatoyac and Malinaltepec, south of Tlapa, all in the state

and produce food products contaminated with parasites” (Slifko of Guerrero, as reference localities for T. guerreroensis. Vegetational,

et al., 2000). In addition, social and cultural changes that modify climatic and socioeconomic affinities in the distribution range of T.

Fig. 4. Alignment of the sequence corresponding to the 28 rRNA amplicon. The nucleotide sequence was analyzed in the NCBI web site. A 100% identity match (192/192)

was obtained between the sample sequence and Paragonimus mexicanus (Subject) 28S ribosomal RNA gene, partial sequence reported in GenBank (HM172619.1).

98 J. Vargas-Arzola et al. / Acta Tropica 137 (2014) 95–98

mexicanus. On other hand, the techniques used in this study (stain-

ing and molecular identification) demonstrate the presence of P.

mexicanus in Putla, western Oaxaca, this location also being a new

report. People living in this community are poor and are primar-

ily engaged in farming and traditional use of natural resources.

Although the prevalence of P. mexicanus was relatively low, it

should nonetheless be considered a potential risk zone for acquiring

paragonimiasis.

Components of local fauna and other environmental factors,

together with the diet of local inhabitants, favor the establishment

Fig. 5. Tehuana guerreroensis, frontal región and orbital región of caparace.

of Paragonimus spp. These conditions suggest that the study region

might be at risk of Paragonimus spp. outbreaks, and identifies this

parasite as a potential target in preventive public health campaigns.

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