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Acta Tropica 137 (2014) 95–98

Contents lists available at ScienceDirect

Acta Tropica

jo ur nal home p age: www.elsev ier .com/ locate /ac ta t ropica

etection of Paragonimus mexicanus (Trematoda) metacercariae inrabs from Oaxaca, Mexico

aime Vargas-Arzolaa, Aristeo Segura-Salvadora, Leobardo Reyes-Velascoa,ylan L. Díaz-Chiguerb, Adrián Márquez-Navarroc, Gloria León-Avilac,abriela Ibanez-Cervantesd, Alejandro D. Camachoc, Rosa Ma. Sánchez-Manzanoc,enjamín Nogueda-Torresc,∗

Facultad de Ciencias Químicas, Universidad Autónoma “Benito Juárez” de Oaxaca, Oaxaca 68120, MexicoInstituto de Seguridad y Servicios Sociales para los Trabajadores del Estado, ISSSTE, México DF 06720, MexicoEscuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, México DF 11340, MexicoCentro Nacional de la Transfusión Sanguínea, México DF 07360, Mexico

r t i c l e i n f o

rticle history:eceived 4 December 2013eceived in revised form 3 May 2014ccepted 9 May 2014

a b s t r a c t

Metacercariae of Paragonimus mexicanus were collected in crabs Tehuana guerreroensis (Rathbun, 1933)in the municipality of Putla, Oaxaca, Mexico. Metacercariae were found in 20.8% of the crabs collected,with an average of 1.9 metacercarie per crab. Stained metacercariae showed the specific characteristicsof P. mexicanus by morphology and sequencing a fragment of the 28S ribosomal gene obtained by PCR.

vailable online 16 May 2014

eywords:aragonimus mexicanusetacercariae

rabs

These findings reveal that T. guerreroensis is an intermediate host for P. mexicanus; this new report isrelevant considering the potential risk of transmission in the states of Oaxaca and Guerrero, Mexico.

© 2014 Elsevier B.V. All rights reserved.

ehuana

. Introduction

Paragonimiasis is a food-borne parasitic disease of humans,ther mammals, molluscs and crustaceans; it is caused by flukes ofhe genus Paragonimus spp. Human paragonimiasis is distributedcross three continents: South and Central America, East Asia andest Africa. Typical symptoms of human paragonimiasis include

ever, cough, eosinophilia and hemoptysis, which together can beisdiagnosed as tuberculosis. Praziquantel is the drug of choice for

aragonimiasis; however, other drugs have been used (Keiser andtzinger, 2010). Paragonimus spp. use snails as first intermediateosts and decapod crustaceans (crabs and freshwater shrimp) asecond intermediate hosts.

Infection occurs when man ingests either raw or uncookedrustaceans infected with metacercariae or meat from paratenic

osts. More than 30 species of the genus Paragonimus have beenescribed; of these, 10 species have been reported to infectumans. Paragonimus mexicanus is located in México, Colombia,

∗ Corresponding author. Tel.: +52 5557296000x62399.E-mail addresses: bnogueda@yahoo.com, bnogueda@hotmail.com

B. Nogueda-Torres).

ttp://dx.doi.org/10.1016/j.actatropica.2014.05.004001-706X/© 2014 Elsevier B.V. All rights reserved.

Perú, Ecuador, Costa Rica, Panamá and Guatemala. In México,this parasite has been found in wild animals from Colima, Chia-pas, Hidalgo, Michoacán, Nayarit, Puebla, San Luis Potosi, Tabasco,Yucatán, Veracruz and State of México (Lamothe-Argumedo, 1985).Several crustacean species of the family Pseudothelphusidae andone of the family Trichodactylidae serve as second intermediatehosts (Blair et al., 1999).

At least 13 genera and 48 species of the family Pheudothel-phusidae have been described in México, as summarized elsewhere(Rodríguez and Magalhães, 2005). P. mexicanus metacercariaehave been found in crustaceans such as Pseudothelphusa dilatata(Lamothe-Argumedo et al., 1977), Ptychophallus tristani and P.costaricensis (Brenes et al., 1980; Mongue et al., 1985), Odontothel-phusa maxillipes and Raddaus tuberculatus (Lamothe-Argumedo,1984), Pseudothelphusa americana belliana, P. nayaritae and P. ter-restris (Lamothe-Argumedo, 1995), all of which serve as secondintermediate hosts. In many regions of Mexico, as in the state ofOaxaca, 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 andevaluate the presence of P. mexicanus from crabs collected in thestate of Oaxaca, Mexico.

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

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Fig. 1. Map of Mexico country and localization

. Materials and methods

.1. Source of crabs

Crabs were collected at San Miguel Reyes, municipality of Putlailla de Guerrero, State of Oaxaca, Mexico (97◦55′45′ ′ West and,7◦01′33′ ′ North at an altitude of 783 m.a.s.l.) (Fig. 1). A total of 120rabs (Fig. 2) of various sizes were collected between February 16thnd March 13th 2011, in small freshwater streams. Water tempera-ure at the time of capture was 21 ◦C; pH was 7.0. Crabs were placedn plastic containers and transported alive to the laboratory.

.2. Isolation of metacercariae

In the laboratory, crabs were allowed to acclimatize for two daysnd then weighed and measured with a caliper. Afterwards, crabsere killed by thermal shock (2 ◦C for 10 min) fixed in 5% formalin

nd then placed in plastic containers. Crabs were then dissected:hey were cut in half from the cephalothorax in longitudinal sec-ion in order to extract the gills, digestive system and ontocele.his material was placed in Petri dishes with saline, the tissue wasnelly sliced with scalpel and carefully examined under a stereo

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

ariae were rinsed in saline and preserved in 96% ethanol until used.ndividual metacercaria were observed under the microscope.

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

tla Villa de Guerrero, state of Oaxaca, Mexico.

2.3. DNA extractions from metacercariae

DNA was extracted from approximately 5 mg of metacercariae.DNA was purified using the DNeasy Blood & Tissue Kit (Qiagen,Valencia, CA, USA) and its concentration was measured using ananoDrop 2000 (Thermo Scientific Waltham MA, USA). DNA fromuninfected crabs maintained and bred in the laboratory was usedas negative control. All DNA samples were stored at −20 ◦C untiluse.

2.4. PCR assay

The DNA extracted was amplified in a 25-�l reaction wellusing 100 ng of DNA template, 0.8 �M each of forward and reverseprimers (final concentration), and Master Mix (Roche). Amplifi-cation was run in a Tc-3000 (TECHNE)®. Cycling was performedas follows: initial DNA denaturing (94 ◦C, 5 min), followed by 30cycles each of denaturing (92 ◦C, 30 s), annealing (61 ◦C, 30 seg),extension (72 ◦C, 1 min). The final extension was 72 ◦C for 4 min.The primers 28S-F 5′-GAGGGTGAAAGGCCCGTGGG-3′ and 28S-R5′-ACGCATGCACACACCTCRAGCCG-3′ were designed in a conserva-tive region bracketing a variable region of approximately 630 bp ofthe 28 S rRNA. Amplicons obtained were analyzed in 1.5% agarosegel stained with ethidium under UV light. Positive and negativecontrols were always included.

2.5. Sequencing and BLAST

The PCR product was sequenced in both strands in the Unitof Proteogenomics, UNAM, Juriquilla, Mexico. The sequences wereviewed in the Chromas program Lite and refined manually. The finalsequence 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 genuslevels based on external morphological features using a NikonSMZ1000 stereo microscope. Males were dissected and the gono-

pod was extracted to allow the specific identification by taxonomickeys (Rodríguez and Smalley, 1696; Rodríguez, 1982). The updatedspecies name was taken from criteria of Alvarez and Villalobos(1994).

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

Table 1Prevalence of Paragonimus metacercariae in crabs Tehuana guerreroensis in a com-munity of Putla Villa de Guerrero, state of Oaxaca, Mexico.

No. of crabs Average ofmetacercariae/crab

No. ofmetacercariaedetected

3

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temmaclb

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Examined Infected

120 25 (20.8%) 1.9 48

. Results

Of a total of 120 crabs examined, 25 specimens (20.83%) wereositive for Paragonimus spp. metacercariae (Table 1). A total of 48etacercariae were found, averaging 1.9 metacercariae per host

range: 1–6 metacercariae per host). Metacercariae were mainlyocated in the digestive system, ontocele and gills.

Individual metacercariae were stained and the internal struc-ures were observed. As seen in Fig. 3, the oral sucker,xcretory system, acetabulum and blind gut showed typicalorphological characteristics of the Paragonimus genus. Theetacercariae were oval (890 ± 195 × 418 ± 94 �m) covered with

spinosed tegument; oral sucker is subapical (93 ± 17 �m) andentral acetabulum (diameter 66 ± 12 �m); pharynx rectangu-ar (110 ± 12.3 × 123 ± 12.3 �m, base) and intestinal ceca endinglindly and extended to the end of the body.

The identification of P. mexicanus was confirmed by sequencing fragment of the 28S ribosomal gene (Fig. 4).

In Figs. 5 and 6 we show the caparace and left gonopod that allowhe identification of crabs. We determined that crabs belong toehuana guerreroensis (Rathbun, 1933) (Crustacea, Brachyura, Pseu-othelphusidae) (= Pseudothelphusa guerreroensis Rathbun, 1933).

. Discussion

Foodborne trematode infections are still an emerging pub-ic health issue. These are closely related to the proximity ofuman settlements to freshwater bodies. Worldwide, an estimated50 million people are at risk of infections with foodborne trema-odes, and at least 292.8 million humans are specifically at risk ofnfection with the lung fluke Paragonimus spp. (Keiser and Utzinger,005, 2009). This parasite can cause zoonosis in humans wheneat infested with it is consumed raw or undercooked. Although

hese parasites occur in Southeast Asia and Western Pacific regionsue to aquaculture and traditional cooking practices, some of themay emerge in other continents through international trade and

mproved transportation and distribution systems (Dorny et al.,

009). It has been proposed that “the rising demands on naturalesources increase the likelihood of encountering environmentsnd produce food products contaminated with parasites” (Slifkot al., 2000). In addition, social and cultural changes that modify

ig. 4. Alignment of the sequence corresponding to the 28 rRNA amplicon. The nucleotias obtained between the sample sequence and Paragonimus mexicanus (Subject) 28S rib

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.

the diet have led humans to explore and in many cases alter themicro- and macro-environmental conditions resulting in the emer-gence or re-emergence of parasitic zoonosis (Macpherson, 2005).The implementation of common parasitological techniques sup-ported by molecular methods such as PCR and DNA sequencing maybe useful in the identification of parasites from different sources,including local fauna, involved in the transmission of parasites.

Identification of P. mexicanus infecting crabs T. guerreroensisfrom the community of San Miguel Reyes, Municipality of PutlaVilla de Guerrero, in the State of Oaxaca, Mexico, lead us to con-sider this crab species as a second intermediate host. The site ofPutla is close to the border with the state of Guerrero. Rodríguez and

Smalley (1696) and Alvarez and Villalobos (1994), report the locali-ties of Copanatoyac and Malinaltepec, south of Tlapa, all in the stateof Guerrero, as reference localities for T. guerreroensis. Vegetational,climatic and socioeconomic affinities in the distribution range of T.

de sequence was analyzed in the NCBI web site. A 100% identity match (192/192)osomal RNA gene, partial sequence reported in GenBank (HM172619.1).

98 J. Vargas-Arzola et al. / Acta Tr

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

ga

rpi

Fig. 6. Tehuana guerreroensis. Left gonopode.

uerreroensis allow us to consider this area of southwestern Mexicos a potential focus of paragonimiasis.

The DNA sequence obtained has a 100% match with the sequenceeported for P. mexicanus. Additionally, it has 98% match with P.seudoheterotremus and P. heterotremus, both species distributedn Asia; this distributional data corroborates our identification of P.

opica 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 newreport. 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, itshould nonetheless be considered a potential risk zone for acquiringparagonimiasis.

Components of local fauna and other environmental factors,together with the diet of local inhabitants, favor the establishmentof Paragonimus spp. These conditions suggest that the study regionmight be at risk of Paragonimus spp. outbreaks, and identifies thisparasite as a potential target in preventive public health campaigns.

References

Alvarez, F., Villalobos, J.L., 1994. Two new species and one new combination of fresh-water crabs from Mexico (Crustacea: Brachyura: Pseudothelphusidae). Proc.Biol. Soc. Wash. 107, 729–737.

Blair, D., Xu, Z.B., Agatsuma, T., 1999. Paragonimiasis and the genus Paragonimus.Adv. Parasitol. 42, 113–222.

Brenes, R.R., Zeledon, R., Rojas, G., 1980. Biological cycle and taxonomic position ofa Costa Rican Paragonimus and the present status of Paragonimus in the newworld. Brenesia 18, 353–366.

Dorny, P., Praet, N., Deckers, N., Gabriel, S., 2009. Emerging food-borne parasites.Vet. Parasitol. 163, 196–206.

Keiser, J., Utzinger, J., 2005. Emerging foodborne trematodiasis. Emerg. Infect. Dis.11, 1507–1514.

Keiser, J., Utzinger, J., 2009. Food-borne trematodiases. Clin. Microbiol. Rev. 22,466–483.

Keiser, J., Utzinger, J., 2010. The drugs we have and the drugs we need against majorhelminth infections. Adv. Parasitol. 73, 197–230.

Lamothe-Argumedo, R., Caballero-Deloya, J., Lázaro-Chávez, M.E., 1977. Parathel-phusa (P.) dilatata Rathbun (Crustacea: Decapoda), segundo hospederointermedio de Paragonimus mexicanus (Trematoda). An. Inst. Biol. UNAM Zool.48, 295–298.

Lamothe-Argumedo, R., 1985. La paragonimiasis en el Continente Americano. Sal.Pub. Mex. 27, 514–523.

Lamothe-Argumedo, R., 1984. Nuevos datos sobre la distribución geográfica deParagonimus mexicanus, y tres nuevos hospederos intermediarios en México.An. Inst. Biol. UNAM Zool. 55, 85–89.

Lamothe-Argumedo, R., 1995. Sobre dos hospederos nuevos de Paragonimus mexi-canus en México. An. Inst. Biol. UNAM Zool. 66, 147–150.

Macpherson, C.N., 2005. Human behaviour and the epidemiology of parasiticzoonoses. Int. J. Parasitol. 35, 1319–1331.

Mongue, E., Brenes, R., Munóz, G., 1985. Infección natural de Phychopallus tristani(Crustacea: Decapoda) con metacercarias de Paragonimus mexicanus (Trema-toda) en Tabarcia de Mora, Costa Rica. Rev. Inst. Med. Trop. Sao Paulo 27, 23–26.

Rodríguez, G., Magalhães, C., 2005. Recent advances in the biology of the Neotropicalfreshwater crab family Pseudothelphusidae (Crustacea, Decapoda, Brachyura).Rev. Bras. Zool. 22, 354–365.

Rodríguez, G., Smalley, E., 1696. Los cangrejos de agua dulce de México de la familiaPseudothelphusidae (Crustacea, Brachyura). Anales Inst. Biol. Univ. Nac. Autón.

Mex. Ser Cienc del Mar y Limnol. 40, 69–112.

Rodríguez, G., 1982. Les crabs d’eau douce d’Amerique, Famille des Pseudothelphusi-dae. Faune. Trop. 22, 1–223.

Slifko, T.R., Smith, H.V., Rose, J.B., 2000. Emerging parasite zoonoses associated withwater and food. Int. J. Parasitol. 30, 1379–1393.