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Journal of Invertebrate Pathology xxx (2007) xxx–xxx

INVERTEBRATE

PATHOLOGY

First record of a nematode Metastrongyloidea (Aelurostrongylusabstrusus larvae) in Achatina (Lissachatina) fulica

(Mollusca, Achatinidae) in Brazil

Silvana C. Thiengo a,*, Monica A. Fernandez a, Eduardo J.L. Torres b, Pablo M. Coelho a,Reinalda M. Lanfredi b

a Laboratorio de Malacologia, Instituto Oswaldo Cruz/Fiocruz, Av. Brasil 4365, Manguinhos 21.040-900, Rio de Janeiro, RJ, Brazilb Laboratorio de Biologia de Helmintos Otto Wucherer, Instituto de Biofısica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro,

Ilha do Fundao 21.949-900, Rio de Janeiro, RJ, Brazil

Received 19 June 2007; accepted 23 October 2007

Abstract

Achatina (Lissachatina) fulica was introduced in Brazil in the 1980s for commercial purposes (‘‘escargot’’ farming) and nowadays,mainly by human activity, it is widespread in at least 23 out of 26 Brazilian states and Brasılia, including the Amazonian region andnatural reserves, where besides a general nuisance for people it is a pest and also a public health concern, since it is one of the naturalintermediate host of Angiostrongylus cantonensis, ethiological agent of the meningoencephalitis in Asia. As Brazil is experiencing theexplosive phase of the invasion, the Laboratorio de Malacologia do Instituto Oswaldo Cruz/Fiocruz has been receiving samples of thesemolluscs for identification and search for Angiostrongylus cantonensis and Angiostrongylus costaricensis larvae. While examining samplesof A. fulica different nematode larvae were obtained, including Aelurostrongylus, whose different species are parasites of felids, dogs, pri-mates, and badger. Morphological and morphometric analyses presented herein indicated the species Aelurostrongylus abstrusus, as wellas the occurrence of other nematode larvae (Strongyluris-like) found in the interior of the pallial cavity of A. fulica. This is the first reportin Brazil of the development of A. abstrusus infective larvae in A. fulica evidencing the veterinary importance of this mollusc in the trans-mission of A. abstrusus to domestic cats. Since the spread of A. fulica is pointed out in the literature as one of the main causative spreadof the meningoencephalitis caused by A. cantonensis the authors emphasize the need of sanitary vigilance of snails and rats from vulner-able areas for A. cantonensis introduction as the port side areas.� 2007 Elsevier Inc. All rights reserved.

Keywords: Aelurostrongylus abstrusus; Angiostrongylus spp.; Achatina fulica; Invasive molluscs; Brazil

1. Introduction

Also known as the Giant African snail, Achatina fulica

Bowdich, 1822 has been introduced throughout the tropicsand subtropics and has been considered the most impor-tant snail pest in these regions. In Brazil it has been intro-duced in 1980s for commercial purposes (‘‘escargot’’farming) that were not successful. Nowadays, mainly byhuman activity, A. fulica is widespread in at least 23 out

0022-2011/$ - see front matter � 2007 Elsevier Inc. All rights reserved.

doi:10.1016/j.jip.2007.10.010

* Corresponding author. Fax: +55 21 2560 2357.E-mail address: sthiengo@ioc.fiocruz.br (S.C. Thiengo).

Please cite this article in press as: Thiengo, S.C. et al., First record otebr. Pathol. (2007), doi:10.1016/j.jip.2007.10.010

of 26 Brazilian states and the Federal capital Brasılia,including the Amazonian region and natural reserves,where it is a pest in ornamental gardens, vegetable gardens,and small-scale agriculture (Thiengo et al., 2007).

Achatina fulica is also a public health concern since it isintermediate host of the Metastrogyloidea nematode Angi-

ostrongylus cantonensis (Chen, 1935), which causes eosin-ophillic meningoencephalitis in humans, notable caseshaving been reported in some Asian countries and Pacificislands (Alicata, 1991; Prociv et al., 2000). It is also apotential host of Angiostrongylus costaricensis (Moreraand Cespedes, 1971), which causes abdominal angiostron-

f a nematode Metastrongyloidea (Aelurostrongylus ..., J. Inver-

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gylosis, a zoonosis that occurs from the southern UnitedStates to northern Argentina. In Brazil, most cases of thisinfection are concentrated in the southern states and manyspecies of terrestrial molluscs have been identified as vec-tors (Graeff-Teixeira et al., 1993).

The genus Aelurostrongylus comprises species parasiticof domestic and sylvatic felids and dogs besides primatesand badgers. The life cycle of Aelurostrongylus abstrusus

(Railliet, 1898) involves two hosts. The eggs are depositedby the females in the smaller branches of the pulmonaryarteries, they are arrested by the capillaries to the lung tis-sue where first stage larvae develop, hatche and break intothe alveoli, go up the bronchiole, bronchi, and trachea, areswallowed and pass out in the faeces. They live as long as 7weeks in water but must invade the snail or slug tissue forfurther development. Definitive host is infected by theingestion of the infected mollusc. Suitable species belongto the Genus: Biomphalaria, Helix, Agriolimax, Subulina,Arion, and Helicella, among others. In the mollusc, larvae

Fig. 1. Map of Brazil showing the municipalities with samples

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molt twice in around 20 days. Various frogs, toads, birds,rodents, lizards, and snakes eat the infected mollusc andcan act as a transport (parathenic) host. In these hosts thirdstage larvae encyst, mainly in the intestinal ligament, last-ing viable for around 12 weeks. Definitive hosts becomeinfected by eating the mollusc or the parathenic host. Lar-vae emerge in the stomach, pass via the blood stream to thelungs where they molt twice becoming adult in 15 days.Larvae pass in faeces in around 39 days (Hamilton andMcCaw, 1968; Soulsby, 1987; Ribeiro and Lima, 2001).In Brazil A. abstrusus have been recorded in domestic catsin several states (Castro et al., 1999; Ribeiro and Lima,2001; Scofield et al., 2005).

Since Brazil is currently experiencing the explosive phaseof the invasion with big specimens of A. fulica occurring indense populations in urban areas, the Laboratory of Mal-acology of Instituto Oswaldo Cruz, as a National Centerfor Medical Malacology has been examining samples ofA. fulica sent by the Brazilian health authorities and envi-

of Achatina fulica examined for Metastrongylidae larvae.

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ronmental agencies in order to determine the health risksfor human population and veterinary implications. Whileexamining those samples different nematode larvae wereobtained and morphological and morphometric analyseswere performed to determine the species. This is the firstreport of the development of A. abstrusus infective larvaefrom several localities in Brazil, as well as the occurrenceof other nematode larvae (Strongyluris-like) found in theinterior of the pallial cavity of A. fulica evidencing the vet-erinary importance of this mollusc in the transmission of A.

abstrusus to domestic cats.

2. Materials and methods

The technique of artificial digestion (Wallace andRosen, 1969) was used for the examination of 3806 speci-mens of A. fulica received in Laboratorio de Malacologiado Instituto Oswaldo Cruz from nine Brazilian states fromMarch, 2002 to February, 2007. In 2002 and 2003 A. fulica

were examined only from the state of Rio de Janeiro: 230and 262 molluscs, respectively. In the subsequent yearsthe examined molluscs were: 445 in 2004 (Espırito Santo,Minas Gerais, and Rio de Janeiro states), 969 in 2005(Espırito Santo, Goias, Mato Grosso, Minas Gerais,Parana, Rio de Janeiro, and Sao Paulo), 1869 in 2006

Table 1Infection of Achatina fulica by Aelurostrongylus abstrusus larvae and Strongylu

states

Year State Municipality Aeluros

Numbe

2002 Rio de Janeiro Niteroi —

2003 Rio de Janeiro Duque de Caxias 1Niteroi —Rio de Janeiro —Sao Goncalo —

2004 Rio de Janeiro Rio de Janeiro —

2005 Espirito Santo Guarapari 5Itaguacu —Serra —Vitoria 1

Goias Jaragua 7Uruacu 5

Rio de Janeiro Mangaratiba —Rio de Janeiro —Sao Goncalo 12

2006 Goias Jaragua 45Niquelandia 81Uruacu 2

Mato Grosso Sao J. Quatro Marcos 3Rio de Janeiro Campos dos Goytacazes 39

Marica —Sao Goncalo 5

Sao Paulo Jundiaı 3Sergipe Lagarto 1

2007 Minas Gerais Paracatu 5Rio de Janeiro Sao Goncalo 1

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(Ceara, Espırito Santo, Goias, Mato Grosso, Rio deJaneiro, Sao Paulo, and Sergipe) and 31 molluscs in 2007(Minas Gerais and Rio de Janeiro) (Fig. 1).

The cephalopodal mass of A. fulica specimens were indi-vidually minced and digested with pepsin (4 mg%) in a0.7% HCl solution for 2 h at 37� C. The digested sampleswere placed in a Baermann apparatus and allowed to sed-iment for 6 h prior to examination. The viscera and the pal-lial organs of the molluscs were placed in Petri dishes withRinger solution and, as well as the sediment collected fromthe bottom of the Baermann funnels, were examined underthe stereomicroscope for helminth larvae.

For light microscopy collected larvae were washed twicein saline to remove tissue debris and fixed in hot AFA (2%glacial acetic acid, 3% formaldehyde, and 95% of 70� etha-nol), clarified and mounted as temporary slides in lactoph-enol solution and examined under an Olympus lightmicroscope. The drawings for the morphometric analyseswere made under a Zeiss Standard microscope with theaid of a camera lucida. For scanning electron microscopylarvae were post-fixed in 1% OsO4 and 0.8% K3Fe (CN)6,dehydrated in graded ethanol (50–100� GL), critical pointdried in CO2, mounted on stubs, coated with gold andexamined under a scanning electron microscope JeolJSM-5310. Measurements were recorded in micrometers.

ris-like larvae from March, 2002 to February, 2007, from seven Brazilian

trongylus abstrusus Strongyluris-like

r infected % infected Number infected % infected

— 5 9.61

3.70 — —— 2 3.07— 1 4.34— 1 5.55

— 17 9.88

9.09 — —— 2 11.76— 1 3.841.44 — —6.14 2 1.755.20 10 10.41— 1 100— 1 1.9628.57 — —

72.58 — —75 — —3.38 3 5.0830 — —3.04 — —— 1 6.668.06 — —6.81 — —33.33 — —

25 — —9.09 — —

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Identification of the nematodes were based on morpho-logical and morphometric parameters following Ash(1970).

Representative specimens of A. abstrusus larvae weredeposited in the Helminthological Collection of theOswaldo Cruz Institute n� 35522 (wet material).

3. Results

From 3806 specimens of A. fulica examined, 216 (5.57%)were infected with A. abstrusus, 43 (1.13%) with Strongylu-

ris-like and from these four snails (0.10%) presented con-comitant infection. Infection indexes by state andmunicipality are presented in Table 1.

The morphometric parameters of the second and thirdlarval stages (L2 and L3) of A. abstrusus obtained by lightmicroscopy are shown in Fig. 2 and the scanning electronmicroscopy of the L3 is shown in Fig. 3. The measurementsof A. abstrusus larvae were compared to those obtained byLopez et al. (2005) (Table 2).

Fig. 2. Aelurostrongylus abstrusus recovered from Achatina fulica by light micregion of infective larvae. (D) Tail of the infective larvae. PR, Posterior regio

Please cite this article in press as: Thiengo, S.C. et al., First record otebr. Pathol. (2007), doi:10.1016/j.jip.2007.10.010

Among the other nematode larvae found in the speci-mens of A. fulica is Strongyluris-like, first reported insnails by Thiengo (1995a). These larvae were found insnails from 10 municipalities in three states: Rio deJaneiro (Angra dos Reis, Mangaratiba, Marica, Niteroi,Rio de Janeiro, and Sao Goncalo municipalities), Goias(Jaragua and Uruacu) and Espırito Santo (Itaguacuand Serra). During examination of the pallial cavity ofA. fulica from Angra dos Reis, prominent cysts contain-ing a highly coiled larva were observed on the surface ofthe pallial tissues (Fig. 4), similar to those found innative specimens of Megalobulimus sp. as described byThiengo (1995a).

4. Discussion

The recent introduction of A. fulica in Brazil must beregarded with caution by health authorities. The humanactivities in addition to its high reproductive capacity andlack of natural pathogens have led to rapid dispersal even

roscopy. (A) Second stage larvae. (B) Infective stage larvae. (C) Anteriorn. AR, Anterior region. KT, knob like tip tail.

f a nematode Metastrongyloidea (Aelurostrongylus ..., J. Inver-

Fig. 3. Aelurostrongylus abstrusus infective larvae by scanning electron microscopy. (A) Whole body. (B) Anterior end. (C) Posterior end. (D) Anteriorend. (AE) Anterior end; (PE) Posterior end. LL, Lateral line; KT, Knobed tip tail; CS, Cuticular striations; OO, Oral opening.

Table 2Measurements (in lm) of Aelurostrongylus abstrusus larvae

Lopez et al., 2005 Present study

Second stage larvae Third stage larvae Second stage larvae Third stage larvae

Length 490 608 454 (424–519) 583 (546–602)Nervous ring 75 90 68 (60–78) 82 (79–86)Excretory pore 95 109 92 (84–97) 101 (95–103)Oesophagus 172 206 170 (168–172) 206 (198–218)Anusa 39 42 35 (30–36) 41 (39–42)

a Distance from the posterior region.

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among distant regions including the Amazonian. The pres-ent study registered the Metastrongylidae larvae of A.

abstrusus in this mollusc indicating also its potential asintermediate host of other helminths (Nematode and

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Trematode: Digenean) of veterinary importance to domes-tic animals in regions of extensive animal farming.

A. fulica can also become intermediate hosts of parasitesof wild animals becoming a risk for the wild fauna biodi-

f a nematode Metastrongyloidea (Aelurostrongylus ..., J. Inver-

Fig. 4. Interior of the pallial cavity of Achatina fulica showing Strong-

yluris-like cysts on the surface of the pallial tissues.

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versity. In addition, the large numbers of this mollusc inareas of variable human population densities, i.e., farawayand urban populations, increase the changes of emergentzoonoses, in addition to those caused by A. costaricensis

and A. cantonensis.Considering all those aspects, we emphasize the need for

further investigations of the potential intermediate hostcapacity of this mollusc for Brazilian endemic parasite spe-cies of human and veterinary interest besides the sanitaryvigilance of snails and rats from vulnerable areas for A.

cantonensis introduction as the port side areas as it was sta-ted by Thiengo (1995b).

Acknowledgments

We thank Dr. Sonia Barbosa dos Santos from theLaboratorio de Malacologia, Universidade do Estado doRio de Janeiro, for providing the A. fulica that appear inFig. 4. Financial support: CAPES-PROCAD, FAPERJ,CNPq, and Secretaria de Vigilancia em Saude/MS.

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