First report of Angiostrongylus cantonensis (Nematoda: Metastrongylidae) in Achatina fulica (Mollusca: Gastropoda) from Southeast and South Brazil

The rat lungworm Angiostrongylus cantonensis is endemic in several parts of the world (Kliks & Palumbo 1992, Wang at al. 2008). Human eosinophilic meningoencephalitis disease caused by this metastrongylid has been reported in southeast Asia (Tsai et al. 2001), the Pacific Islands (Alicata 1962, Rosen et al. 1967, Slom et al. 2002), North America (Diaz 2008) and South America (Caldeira et al. 2007, Lima et al. 2009, Thiengo et al. 2010). This nematode has been reported to infect several different terrestrial and freshwater snail species as intermediate hosts, including Achatina Lissachatina fulica (Bowdich 1822) and Pomacea canaliculata (Lamarck 1822) (Liu et al. 2007). Rodents, including Rattus norvegicus and Rattus rattus, have been reported as natural definitive hosts in Africa (Yousif & Ibrahim 1978, Foronda et al. 2010), Australia (Mackerras & Sandars 1955, Bhaibulaya 1968), North America (Qvarnstrom et al. 2007, Diaz 2008) and some Asian countries (Lv et al. 2009). A. cantonensis infections in A. fulica have been reported in three Brazilian states: Espírito Santo (ES), São Paulo (Caldeira et al. 2007) and Pernambuco (PE) (Thiengo et al. 2010). In this study, A. fulica specimens were collected by health workers from two municipalities in the state of Rio de Janeiro (Barra do Piraí and São Gonçalo) and one municipality in the state of Santa Catarina (Joinville). These specimens were then examined for parasitism with L3 larvae of A. cantonensis in the Malacology Laboratory of Oswaldo Cruz Institute (IOC). In brief, the molluscs were individually minced and digested in a 0.7% HCl solution for 6 h. The digested samples were then placed in a Baermann apparatus and allowed to sediment overnight. Out of 207 A. fulica snails tested, 44 (21.2%) presented infection with L3 larvae of A. cantonensis alone and 17 (8.2%) presented a concomitant infection with Strongyluris or Rhabditis sp. The prevalences of infection are listed in Table I. The L3 nematode larvae obtained from digested snails were administered orally to 3-month-old R. norvegicus (Wistar strain) rats (100 L3/rodent). Thirty-five days after administration of the larvae, the rodents were euthanized using a CO2 chamber; adult worms were then collected from the pulmonary arteries, washed in physiologic solution and fixed in 2% glacial acetic acid, 3% formaldehyde and 95% ethanol (AFA) at 60oC. Ten male and 10 female specimens from each isolate were cleared and mounted as temporary slides in lactophenol solution and examined under a light microscope; drawings for the morphometric analyses were then made with the aid of a camera lucida. Unless otherwise stated, all measurements are given in millimetres. Taxonomic identification of the nematodes was based on morphological Financial support: CNPq, IOC-FIOCRUZ + Corresponding author: maldonad@ioc.fiocruz.br Received 7 May 2010 Accepted 20 July 2010 First report of Angiostrongylus cantonensis (Nematoda: Metastrongylidae) in Achatina fulica (Mollusca: Gastropoda) from Southeast and South Brazil

In this study, A. fulica specimens were collected by health workers from two municipalities in the state of Rio de Janeiro (Barra do Piraí and São Gonçalo) and one municipality in the state of Santa Catarina (Joinville). These specimens were then examined for parasitism with L 3 larvae of A. cantonensis in the Malacology Laboratory of Oswaldo Cruz Institute (IOC). In brief, the molluscs were individually minced and digested in a 0.7% HCl solution for 6 h. The digested samples were then placed in a Baermann apparatus and allowed to sediment overnight. Out of 207 A. fulica snails tested, 44 (21.2%) presented infection with L 3 larvae of A. cantonensis alone and 17 (8.2%) presented a concomitant infection with Strongyluris or Rhabditis sp. The prevalences of infection are listed in Table I. The L 3 nematode larvae obtained from digested snails were administered orally to 3-month-old R. norvegicus (Wistar strain) rats (100 L3/rodent). Thirty-five days after administration of the larvae, the rodents were euthanized using a CO 2 chamber; adult worms were then collected from the pulmonary arteries, washed in physiologic solution and fixed in 2% glacial acetic acid, 3% formaldehyde and 95% ethanol (AFA) at 60ºC. Ten male and 10 female specimens from each isolate were cleared and mounted as temporary slides in lactophenol solution and examined under a light microscope; drawings for the morphometric analyses were then made with the aid of a camera lucida. Unless otherwise stated, all measurements are given in millimetres. Taxonomic identification of the nematodes was based on morphological Financial support: CNPq, IOC-FIOCRUZ The rat lungworm Angiostrongylus cantonensis is a worldwide-distributed zoonotic nematode that can cause human eosinophilic meningoencephalitis. Here, for the first time, we report the isolation of A. cantonensis from Achatina fulica from two Brazilian states: Rio de Janeiro (specifically the municipalities of Barra do Piraí, situated at the Paraiba River Valley region and São Gonçalo, situated at the edge of Guanabara Bay) and Santa Catarina (in municipality of Joinville). The lungworms were identified by comparing morphological and morphometrical data obtained from adult worms to values obtained from experimental infections of A. cantonensis from Pernambuco, Brazil, and Akita, Japan. Only a few minor morphological differences that were determined to represent intra-specific variation were observed. This report of A. cantonensis in South and Southeast Brazil, together with the recent report of the zoonosis and parasite-infected molluscs in Northeast Brazil, provide evidence of the wide distribution of A. cantonensis in the country. The need for efforts to better understand the role of A. fulica in the transmission of meningoencephalitis in Brazil and the surveillance of molluscs and rodents, particularly in ports, is emphasized. and morphometric parameters obtained from previous studies (Chen 1935, Mackerras & Sandars 1955, Alicata 1963, Yousif & Ibrahim 1978. For comparison, specimens of A. cantonensis from Akita, Japan (Department of Parasitology, Medical School, Akita University, Japan) and A. cantonensis from PE (Thiengo et al. 2010), were also analyzed. Representative adult specimens of A. cantonensis were deposited in the Helminthological Collection of IOC under the following reference numbers: CHIOC 35702 (Barra do Piraí isolate: 2 males and 2 females), CHIOC 35703 (São Gonçalo isolate: 2 males and 2 females) and CHIOC 35704 (Joinville isolate: 1 male and 4 females).
The measurements of adult A. cantonensis are listed in Table II. The bursal rays of the São Gonçalo specimens were longer and thinner than those of the specimens from Japan and PE (Figure). The size of the spicules was very similar among the three isolated specimens (Table  II). The bursal morphological characteristics of the Barra do Piraí specimens more closely matched specimens from Canton (Asia) (Chen 1935) and PE (Thiengo et al. 2010), whereas the São Gonçalo specimens more closely matched A. cantonensis from Africa (Yousif & Ibrahim 1978). The particular characteristics of the caudal bursa and the spicule length allowed us to identify the studied specimens as A. cantonensis. R. norvegicus is considered an exotic species in almost all continents except Asia, where it is native and has been found infected with A. cantonensis. The spread of this nematode to the North American continent has been attributed to the introduction of R. norvegicus carried in containers being transported on ships (Diaz 2008).
Helminthological surveys of wild rodents in South America have shown that sigmodontine rodents are the primary hosts for Angiostrongylus spp in the following cases: Angiostrongylus lenzii in Akodon montensis (Souza et al. 2009), Angiostrongylus morerai in Akodon azarae (Robles et al. 2008) and Angiostrongylus costaricensis in Oligoryzomys nigripes and Sooretamys angouya (syn. O. ratticeps) (Graeff-Teixeira et al. 1990). However, no detailed information regarding the definitive hosts of A. cantonensis in South America is currently available. In Brazil, L 1 larvae of A. cantonensis were isolated from the faeces of naturally infected R. norvegicus in ES (Caldeira et al. 2007), though the adult worm morphology was not described. The wide distribution of A. cantonensis in Brazil is likely a result of multiple introductions of parasite-carrying rats during the country's colonial period, due to active commerce with Africa and Asia at that time. Together, the discovery of A. cantonensis in a municipality situated far from the coast in the Paraiba River Valley region and the observed intra-specific morphological variability of adult worms corroborate this hypothesis.
The rapid spread of A. fulica over the country (now having reached 24 out of 26 Brazilian states and the Federal District) is likely contributing to the dispersion of the parasite (Thiengo et al. 2010). This phenomenon is described in the literature as one of the primary causes of the spread of this zoonosis (Kliks & Palumbo 1992, Lv et al. 2009).
Further studies to enhance understanding of the infection dynamics of A. cantonensis in natural vertebrate and invertebrate hosts could help elucidate the role of wild rodents and snails in the transmission of angiostrongyliasis.
The present report of A. cantonensis in Southeast and South Brazil broadens the reported distribution of this parasite in the country. From a public health perspective, it stresses the need for surveillance and control of A. fulica and rodents, particularly in and around ports.