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MORPHOLOGICAL DESCRIPTION AND LIFE CYCLE OF PARAGONIMUSSP. (TREMATODA: TROGLOTREMATIDAE): CAUSAL AGENT OF HUMANPARAGONIMIASIS IN COLOMBIAAuthor(s): Imelda Vélez, Luz E. Velásquez, Iván D. VélezSource: Journal of Parasitology, 89(4):749-755. 2003.Published By: American Society of ParasitologistsDOI: http://dx.doi.org/10.1645/GE-2858URL: http://www.bioone.org/doi/full/10.1645/GE-2858

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749

J. Parasitol., 89(4), 2003, pp. 749–755q American Society of Parasitologists 2003

MORPHOLOGICAL DESCRIPTION AND LIFE CYCLE OF PARAGONIMUS SP.(TREMATODA: TROGLOTREMATIDAE): CAUSAL AGENT OF HUMAN PARAGONIMIASISIN COLOMBIA

Imelda Velez, Luz E. Velasquez, and Ivan D. VelezProgram for the Study and Control of Tropical Diseases–PECET, University of Antioquia, AA 1226, Carrera 50 No. 63-85, Medellın, Colombia.e-mail: ivelez@matematicas.udea.edu.co

ABSTRACT: The first morphological description is made of all stages of the life cycle of a Paragonimus species infecting humansin Colombia. Larval stages were obtained both in vitro and from field collections. Adult Paragonimus spp. are described. Theaquatic snail Aroapyrgus sp. serves as an intermediate host of this species, both naturally and experimentally, yielding rediaeand cercariae. Crabs (Hypolobocera bouvieri monticola and H. emberarum) were found to be the natural second intermediatehosts, and individuals of another crab species (Strengeria sp.) were also infected in the laboratory.

Paragonimiasis is a disease of humans and other mammalscaused by several species of Paragonimus. The parasite com-pletes its life cycle in 2 intermediate hosts, an aquatic snail anda crustacean. Mammals, including humans, become infectedwhen they ingest raw crustaceans containing metacercariae(Yokogawa, 1965, 1969, 1982; Miyazaki, 1982).

More than 40 species of Paragonimus have been describedworldwide, 9 of them from the Americas (see review by Blairet al., 1999). The latter include: P. kellicotti Ward, 1908; P.mexicanus Miyazaki and Ishii, 1968; P. caliensis Little, 1968;P. peruvianus Miyazaki et al., 1969; P. ecuadoriensis Voelkerand Arzube, 1979; P. amazonicus Miyazaki et al., 1973; P.rudis (Diesing, 1850) Stiles and Hassall, 1900; P. napensisAmunarriz, 1991; P. inca Miyazaki et al., 1975; and an un-named Venezuelan species (Tongu et al., 1990; Noya et al.,1992).

In the Americas, the first intermediate hosts of Paragonimusspp. are small snails belonging to the Pomatiopsidae, i.e., Po-matiopsis lapidaria for P. kellikotti (Ameel, 1934), and the Hy-drobiidae, i.e., Aroapyrgus costaricensis and A. alleei for P.mexicanus (Brenes and Rojas, 1975; Lamothe-Argumedo et al.,1977; Lamothe-Argumedo, 1982; Malek et al., 1975); A. col-ombiensis for P. caliensis (Malek and Little, 1971), P. peruvi-anus (Miyazaki et al., 1969), and P. ecuadoriensis; and Aroa-pyrgus sp. for Paragonimus sp. from Venezuela (Tongu et al.,1990). The second intermediate hosts include species of smallcrabs, e.g., Pseudothelphus, Ptychophallus, Psedothelphusa,Potamocarcinus, Strengeria, and Hypolobocera (Lamothe-Ar-gumedo, 1985). The final hosts include wild and domestic cats(Felis spp.), mustelids (Mephitis macrura), canines (Canis sp.),opossums (Didelphis spp., Phillander sp.), pigs, and rodents(Caballero and Montero-Gei, 1961; Brenes et al., 1968). InSouth America, P. mexicanus, P. peruvianus, and P. ecuador-iensis and the Paragonimus sp. from Venezuela have all beenfound to infect humans (Alarcon, Abreu et al., 1985; Alarcon,Noya et al., 1985).

The natural foci of paragonimiasis are located in subtropicaland tropical regions. The habitats include rocky rivulets andsmall creeks whose benthic communities of macroinvertebratesinclude appropriate mollusk and crustacean hosts. Until 1993,only 3 cases of human paragonimiasis had been diagnosed inColombia (Buitrago et al., 1981; Gomez et al., 1984; Restrepo,

Received 30 November 2001; revised 18 February 2003; accepted 18February 2003.

1986); it was not known where or how the patients had acquiredthe parasite for any of these infections (Velez et al., 1995). In1993, the first case from the northwest of Colombia was diag-nosed in a patient belonging to the Embera Indian communityin the municipality of Urrao. Since 2000, 24 more cases havebeen diagnosed (Velez et al., 2000), and the intermediate hostsand different stages of the parasite have been identified. In thepresent article, the results of morphological studies of the Par-agonimus sp. from the Embera area are presented.

MATERIALS AND METHODS

Study area

The study was carried out in the Embera Indian community on the‘‘Valle de Perdidas’’ reservation, situated within the municipality ofUrrao (6832930N, 768209990W), Colombia. The diet of the inhabitants isknown to include raw crabs (Velez et al., 1995). The original vegetationof the region is classified as very humid tropical forest (Holdridge,1987).

Obtaining the different stages of the parasite for morphologicaldescription

The intermediate hosts were collected in creeks used as latrines byEmbera Indians. Snails and crabs were captured alive, taken to the PE-CET Malacology Laboratory, and maintained in aquaria. Eggs werecollected from the sputum in Embera patients and from the adult Par-agonimus sp. dissected from cats that had been exposed to metacercar-iae removed from naturally infected crabs. Dimensions of larval stagesobtained in the field and under experimental conditions were compared.

All parasite stages were observed both live and in permanent prep-arations (except for miracidia). Scanning electron microscope imageswere of eggs, metacercariae, and adults. Immature stages were fixedwith Bouin fixative and subsequently stained with boric carmine, de-hydrated, and then cleared with phenol and methyl salicylate. Entellanwas used as a mounting medium. All measurements are given in mil-limeters.

Adult flukes were obtained from 3 female cats that were experimen-tally infected by oral intubation with 10, 13, or 20 metacercariae. Fivemonths after exposure, eggs were found in the feces of the cats. Dis-section of these cats yielded adult worms.

For the permanent preparations of adult parasites, 30 live specimenswere pressed between 2 slides tied together with thread (B. Werding,pers. comm.) and then fixed at 22 C with FAA (Schell, 1970). Theywere then stained with boric carmine and eosin dissolved in 95% al-cohol, differentiated in 20 ml of distilled water with 5 drops of 8%sodium hypochlorite passed to 95% alcohol, and finally cleared withphenol and methyl salicylate before being mounted on permanent slideswith Entellan. They were observed, measured, and drawn by using astereomicroscope. Five specimens were reserved for future moleculargenetics studies.

750 THE JOURNAL OF PARASITOLOGY, VOL. 89, NO. 4, AUGUST 2003

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VELEZ ET AL.—PARAGONIMUS OF COLOMBIAN EMBERA INDIANS 751

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cyst RESULTS

Eggs were similar to those reported in other species of Par-agonimus. The main characteristics are detailed in Table I. Themiracidia began hatching 19–29 days after eggs were shed. Themiracidium is oval (length 0.083, width 0.031 anteriorly andtapering to 0.021 posteriorly), and its surface is covered bynumerous long cilia. Those of the cephalic region are 0.01 long(Fig. 1). Sporocysts were not observed.

Among the wild snails isolated for cercariae shedding, onlythose Hydrobiidae identified as Aroapyrgus sp. had natural in-fections with Paragonimus sp. Two (1.6%) of 120 snails wereinfected (Velasquez et al., 1999). These snails yielded a totalof 38 rediae from the hepatopancreas. Twenty of the snails thatwere infected in the laboratory and subsequently shed cercariaewere dissected. Between 8 and 32 rediae per snail were found,of which 30 were measured. The rediae are elongated sacs witha well-defined pharynx and contain abundant cercariae with aprominent stylet as in other Paragonimus. Rediae measure-ments are given in Table I.

One to 225 cercariae were shed per mature snail, especiallyin the mornings and early afternoons. Cercariae measurementsare presented in Table II. Spines that are progressively smallertoward the posterior cover the cercariae tegument. The small,rounded tail is somewhat mobile and bears a few posteriorspines that are longer than those of the body. The penetrationglands are situated in 2 lateral groups near the acetabulum, eachhaving 3 lateral cells and 4 smaller medial cells. The excretorybladder is oval, located between the acetabulum and the tail,and opens anterior to the tail. The pseudosucker is a triangularventral depression with the apex toward the tail and thick wallswith prominent edges.

Twenty-two parasitized crabs were found (50%), with an av-erage of 8 metacercariae per animal, all unencysted and mostlying within the intestinal diverticuli of the cephalothorax. Afew metacercariae were also found inside the pleopods of spec-imens of H. bouvieri monticola and H. emberarum (Camposand Rodriguez, 1995). The description of the metacercariae isbased on 25 specimens. Simple spines of various lengths coverthe tegument, except for the orifices of the suckers and the areassurrounding the upper lip of the oral sucker and the acetabulum.The Strengeria crabs infected in vitro were dissected and 56metacercariae recovered.

The metacercariae have a short, muscular, subspherical phar-ynx located behind the oral sucker; adjacent to this can be ob-served the bifurcation of the intestinal cecae, which then zigzagtoward the posterior. The acetabulum is round, muscular, andpostequatorial. Metacercaria measurements are presented in Ta-ble III.

Ten, 13, and 12 adults (100, 100, and 60%), respectively,were recovered from the lungs of the 3 experimentally infectedfemale cats. The adults were found in pairs in lung cysts, exceptfor 1 cyst that had 5 parasites. The measurements were basedon 12 specimens.

The adult parasite has a large, ventrally concave, oval body(Fig. 2), with a thick tegument covered with simple spines. Theoral sucker is round, muscular, and terminal. The pharynx islocated immediately posterior to the oral sucker. Adjacent tothe pharynx is the fork of the intestinal cecae, which are lateral,wide, and zigzag toward the posterior. The acetabulum is round

752 THE JOURNAL OF PARASITOLOGY, VOL. 89, NO. 4, AUGUST 2003

TABLE IV. Morphological comparison of adults from American species (in mm).*

Species Length Width

Oral sucker

Length Width

Paragonimus caliensis Little, 1968P. ecuadoriensis Voelker & Arzube (1979)P. mexicanus Miyazaki & Ishii, 1968P. peruvianus Ibanez et al., 1974Paragonimus sp. (Venezuela) Noya et al., 1992Paragonimus of Embera

7.5–13.36.4–9.3

13.5–14.812.76

8.5–15.0 (10.7)12–16 (15)

4.2–6.43.4–4.26.6–7.96.523.2–7.0 (4.4)6.0–8.0 (7.1)

0.60–1.050.56–0.690.500.690.45–0.76 (0.59)0.70–0.90 (0.78)

0.70–1.200.64–0.900.700.850.57–0.96 (0.80)0.70–1.10 (0.93)

* L, left; R, right.

FIGURE 1. Miracidia of Paragonimus sp.

and muscular, small in relation to body size, and slightly smallerthan the oral sucker. The highly lobed ovary is located centrally,behind the acetabulum. The testes are posterior to the ovary.Vitelline follicles are abundant; they are lateral along the bodyin the form of very small peripheral lobes that surround theintestinal cecae both dorsally and ventrally, from the pharynxto the termination of the intestinal cecae. Transverse vitellineducts are equatorial and meet at the posterior edge of the ovary.The genital pore is immediately posterior to the acetabulum.The uterus, the seminal receptacle, and the metraterm are lo-cated between the acetabulum and the ovary, and the genitalpore can be observed easily. Adult measurements are presentedin Table IV.

Specimens deposited: Holotype: accession no. 090274.00,storage no. MT30-20A.

Paratype: Accession no. 090275.00, storage no. MT30-20B/F; U.S. Department of Agriculture, U.S. National Parasite Col-lection, Maryland.

DISCUSSION

The taxonomy of Paragonimus spp. in the Americas is con-fusing. Species such as P. rudis and P. napensis have beendescribed inadequately, whereas others show few morphologi-cal differences from other members of the genus with respectto some of their stages. An example of this is P. peruvianus,whose metacercariae cannot be differentiated morphologicallyfrom the metacercariae of P. mexicanus (Miyazaki et al., 1971).Some authors (Yokogawa, 1969; Thatcher, 1993) have claimedthat P. mexicanus, P. peruvianus, and P. ecuadoriensis are thesame species because of the similarity in the eggs and in theevolution of the life cycle of these species. However, othersclaim that there are sufficient morphological differences forthem to be considered as distinct species (Ibanez, 1990).

We consider that the specimens obtained from Valle de Per-didas belonged to a single species because we found no differ-ence in the morphology of the eggs obtained from 3 sources

VELEZ ET AL.—PARAGONIMUS OF COLOMBIAN EMBERA INDIANS 753

TABLE IV. Extended.

Acetabulum

Length Width Pharynx

Testes

Length Width

Ovary

Length Width

0.75–1.050.62–0.770.660.760.41–0.86 (0.74)0.50–0.77 (0.66)

0.80–1.050.62–0.750.640.770.39–1.1 (0.77)0.50–0.80 (0.72)

0.51 3 0.3

0.42–0.38 (0.42)

5–6 lobes5–6 lobes, R 2.7, L 1.56

3–6 lobes, R 1.64L 1.38–1.44, R 1.31–1.36

R 1.04, L 0.86

R 0.82L 0.78–1.57, R 0.68–1.50

0.80–1.826 lobes, 0.83–1.46

6–9 lobes, 0.68–1.290.73–0.78

0.60–1.500.71–1.66

0.35–1.020.89–1.13

FIGURE 2. Paragonimus sp. A. Juvenile with a few eggs. B. Adult with numerous eggs. C. Adult with numerous eggs.

(human sputum, feces, and cat feces), the life cycle, or themorphology of the adult forms. Unfortunately, we cannot de-termine whether this Embera isolate represents a new speciesof Paragonimus and provide a formal description because thereare conflicting descriptions of the morphological and life cyclesof the American species of Paragonimus recognized so far (P.mexicanus, P. ecuatoriensis, P. peruvianus, and P. caliensis).

The miracidia released from Paragonimus sp. eggs obtainedfrom members of the Embera community are the first to bedescribed for Central or South American species. Althoughhatching time differed from that of P. mexicanus by approxi-mately 83 days, this could possibly be attributed to the lightstimulus used in our study. No mention is made of a lightsource in the study by Rangel and Lamothe (1986).

Comparative analyses of the different stages of the life cycleindicate that the main interspecific differences involve the cer-

cariae and the adults. Unfortunately, there is little informationon the comparative morphology of miracidia and rediae. Ac-cordingly, we carefully examined these stages and describe forthe first time some morphological details of the miracidia ofthe new Paragonimus sp. in the Western Hemisphere. Althoughwe could not find sporocysts, we found no significant differencebetween the rediae of the Embera Paragonimus sp. and othermembers of the genus (see Table I).

The presence of a pseudosucker on the cercaria is a cleartaxonomical character that was found both in the Embera Par-agonimus sp. and P. mexicanus but is absent from P. peruvi-anus and Paragonimus sp. from Venezuela. Unfortunately, it isnot known whether P. caliensis and P. ecuadoriensis have pseu-dosuckers. Additional morphological characteristics of cercar-iae (Table II) further emphasize the similarity between EmberaParagonimus sp. and P. mexicanus. The Embera metacercariae

754 THE JOURNAL OF PARASITOLOGY, VOL. 89, NO. 4, AUGUST 2003

are the largest of any American species and, as in all otherspecies except P. caliensis, are not encysted.

The adult of the Paragonimus sp. is only slightly larger thanParagonimus sp. of Venezuela and P. caliensis (Table IV). Theoral sucker is almost as large as that described for P. caliensis,which is the largest in South America. The acetabulum of theParagonimus sp., however, is one of the smallest, similar tothose of P. mexicanus and P. ecuadoriensis.

The majority of authors use morphological differences in thegenitalia as taxonomical criteria to differentiate species (Blairet al., 1999). The main difference between the Paragonimus sp.and P. caliensis is the distance between the testes and the pos-terior pole of the body, which is greater in the latter. In addition,the ovary of the Paragonimus sp. is more lobulated, whereasthe uterus is larger and extends past the acetabulum. The maindifference between P. ecuadoriensis and the Paragonimus sp.is that in the former the testes are situated at the same level,whereas the one on the side of the uterus in the latter is posi-tioned somewhat lower than the other.

The main difference between the Paragonimus sp. and P.mexicanus is that the latter has much longer testes that reachthe posterior pole of the body and display a well-defined sym-metry (Lamothe et al., 1978). In addition, the suckers and ovaryof P. mexicanus are larger, and the latter lies almost parallel tothe acetabulum. On the basis of a comparison with a more re-cent description of P. mexicanus (Lamothe-Argumedo, 1985),it can be concluded that the testes of the Paragonimus sp. arelarger overall and that the uterus extends past the acetabulum.Although morphological analyses of the genitalia of Paragon-imus spp. have clearly helped in defining the different species,studies conducted with P. peruvianus indicate that a significantdegree of variability can occur within a single species (Ibanezet al., 1974).

The results presented here suggest that the Paragonimus sp.isolated from members of the Embera community may be anew species. This is supported by the existence of several pe-culiarities in its life cycle and morphological characteristics,which although similar to those of P. mexicanum, are nonethe-less distinct. The parasite is clearly different from other speciesdescribed from the Americas, i.e., P. peruvianus, P. ecuador-ianis, P. caliensis, and Paragonimus sp. from Venezuela. Theresults of the present study also draw attention to the need tocomplement morphological analyses with the use of molecularprobes to define the different species of Paragonimus in theAmericas.

ACKNOWLEDGMENTS

We thank members of the Embera communities of the Colombiandepartments of Antioquia and Choco, D. Blair (Department of Zoologyand Tropical Ecology, James Cook University, Townsville, Australia),M. Aciaga (Chiba University, Japan), C. Naquira and M. Tantalean(University of San Marcos, Peru), Martha Rocha (Instituto de CienciasNaturales, Bogota), Airborne Health Program (Programa Aereo de Sal-ud del Departamento de Antioquia), and the University of Antioquia.

LITERATURE CITED

ALARCON, B., G. ABREU, AND O. NOYA. 1985. Pathological and parasi-tological aspects of the first autoctonous case of human paragoni-miasis in Venezuela. American Journal of Tropical Medicine andHygiene 34: 761–765.

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