The Occurrence of the Rat Lungworm, Angiostrongylus cantonensis, in Nonindigenous Snails in the Gulf of Mexico Region of the United States

Abstract

Nonindigenous apple snails, Pomacea maculata (formerly Pomacea insularum), are currently spreading rapidly through the southeastern United States. This mollusk serves as an intermediate host of the rat lungworm parasite (Angiostrongylus cantonensis), which can cause eosinophilic meningitis in humans who consume infected mollusks. A PCR-based detection assay was used to test nonindigenous apple snails for the rat lungworm parasite in Louisiana, Texas, Mississippi, and Florida. Only apple snails obtained from the New Orleans, Louisiana, area tested positive for the parasite. These results provide the first evidence that Angiostrongylus cantonensis does occur in nonindigenous apple snails in the southeastern United States. Additionally, Angiostrongylus cantonensis was identified in the terrestrial species Achatina fulica in Miami, Florida, indicating that rat lungworm is now established in Florida as well as Louisiana. Although the study suggests that the rat lungworm is not widespread in the Gulf States region, the infected snail population could still pose a risk to human health and facilitate the spread of the parasite to new areas.

Introduction

Pomacea maculata (formerly referred to as Pomacea insularum, the island apple snail, which is now a junior synonym)¹ is a large freshwater snail species native to South America that has been introduced to the southeastern United States. Established populations currently occur in Texas, Louisiana, Alabama, Florida, Georgia, South Carolina, and North Carolina.^2,3 This species can serve as a host for the rat lungworm, Angiostrongylus cantonensis,⁴ a parasite that can cause disease in people who consume infected mollusks. Angiostrongylus cantonensis is established in Southeast Asia, Australia, the Pacific Islands, Caribbean, and more recently in the Americas, where it is the causative infective agent for eosinophilic meningitis, a serious parasitic infection of humans.^5–7 Previous studies detected the rat lungworm in rats in New Orleans⁸ and in mammalian accidental hosts in the proximity of New Orleans.^9,10 In Florida, A. cantonensis was found in an infected gibbon at the Miami Metro Zoo.¹¹ These results suggest that P. maculata in these areas might serve as an intermediate host for A. cantonensis and facilitate the spread of the parasite as the snail's range continues to expand through the region.

Recently P. maculata have been observed in the canals of New Orleans where they are reportedly harvested for consumption by various ethnic groups, putting those groups at potential risk for infection with the parasite (John Palmisano, West Bank Drainage District, oral communication to John Teem, June 2007). The extent to which infections of rat lungworm occur in P. maculata populations in New Orleans remains unknown. In addition, there have been no studies to determine whether rat lungworm infections occur in P. maculata populations in Miami or in any of the other areas where P. maculata occurs in the states of the Gulf of Mexico region. It is further unknown whether other invasive snail species in the region are presently infected with A. cantonensis.

To determine the extent to which P. maculata populations within the region are infected with rat lungworm, samples were gathered from sites in Louisiana, Texas, Mississippi, and Florida and adult snail tissue analyzed for the presence of rat lungworm using PCR-based detection assays.¹² The extent to which Achatina fulica, a nonindigenous terrestrial snail species recently introduced to Florida, is infected with the parasite was also investigated.

Methods

From each location sampled (Table 1, Figure 1), nonindigenous apple snails were collected and frozen at −20°C. Using a sterile disposable scalpel and forceps, a section of the posterior portion of the foot muscle approximately 1 cm in length and 0.5 cm in diameter was cut from each snail and these samples were then frozen at −20 °C until processed for DNA extraction.

Table 1.

Results of PCR-based detection of A. cantonensis in apple snail samples collected in the southeastern United States.

PM - Pomacea maculata, MC - Marisa cornuarietis, AF - Achatina fulica.

Location			Lat./Long.	No. of samples	No. of positives	Species
1	Houston, Texas	Rice/crawfish farm	29 22 13.30 N 95 26 09.35 W	60	0	PM
2	Schriever, Louisiana	Swamp and bayou	29 43 45.51 N 90 51 21.25 W	40	0	PM
3	Gretna, Louisiana	Verret Canal	29 52 37.25 N 90 02 36.37 W	60	8	PM
4	Mandeville, Louisiana	Stormwater retention pond, drainage ditch	30 22 48.01 N 90 02 24.37 W	40	1	PM
5	Picayune, Mississippi	Residential community lake	30 33 48.35 N 89 38 52.40 W	42	0	PM
6	Everglades National Park, Florida	Tamiami Canal	25 45 43.91 N 80 45 59.10 W	52	0	PM
7	Miami, Florida	Miami Metro Zoo canal	25 36 33.47 N 80 23 52.37 W	52	0	MC
8	Miami, Florida	Miami residential areas	25 40 18.30 N 80 25 42.00 W	140	4	AF

Figure 1.

Geographic location of apple snail sampling sites (circles) in the southeastern United States in relation to watersheds where P. maculata populations have become established (shaded). Black circles and triangles indicate locations in which apple snails and giant African land snails (Achatina fulica), respectively, tested positive for rat lungworm. 1. Houston, Texas, 2. Schriever, Louisiana, 3. Gretna, Louisiana, 4. Mandeville, Louisiana, 5. Picayune, Mississippi, 6. Everglades National Park, Florida, 7. Miami Metro Zoo, Miami, Florida, 8. Residential Community, Miami, Florida. Map source USGS Nuisance Aquatic Species, http://nas3.er.usgs.gov/

Total cellular DNA (tDNA) was extracted from approximately 0.1 g of foot tissue from each snail using the DNeasy blood and tissue kit (Qiagen) or Idaho Technologies 1-2-3 DNA purification kit following the manufacturers' protocols. The presence of A. cantonensis in the snail samples was detected by a real-time PCR assay.¹² Samples of Achatina fulica were also assayed by nematode extraction and identification methods that will be detailed elsewhere (Smith, unpublished). A subset of samples that tested positive was selected for confirmation of infection using a conventional PCR assay to amplify and sequence the 18S rRNA gene. Agarose gel electrophoresis was used to separate the resulting DNA fragments, which were sequenced to confirm the nematode species identity.¹³

Results

Pomacea maculata occurs in a large proportion of the watersheds in Florida, including the Miami area where a gibbon infected with A. cantonensis was identified previously at the Miami Metro Zoo.¹¹ The Miami Metro Zoo contains numerous canals and ponds that could potentially support P. maculata, yet no P. maculata were found in any of them. However, another nonindigenous apple snail, Marisa cornuarietis (the giant ramshorn snail) was found in a pond near the exhibit that once housed the gibbon infected with A. cantonensis.¹¹ Angiostrongylus cantonensis infects a broad range of mollusk species, including M. cornuarietis.¹⁴ Accordingly, samples were taken from this Marisa cornuarietis population for testing. All 52 snails from the Miami Metro Zoo tested negative for the parasite using the PCR-based methods (Table 1), as did the P. maculata from a population within the nearby Tamiami Canal (north of Everglades National Park). These results indicate that rat lungworm is not widespread among apple snail populations in the area. However, there is another invasive snail species recently detected in the Miami area, which could also serve as a new reservoir for the parasite. The giant African land snail, Achatina fulica, is a nonindigenous terrestrial snail discovered at multiple residential locations in the Miami area in September 2011. Achatina fulica serves as a reservoir for rat lungworm in other areas of the world where it has been introduced.^5,15 Therefore, samples of Achatina fulica were tested from 12 of the 19 core areas where A. fulica has been found in the Miami area. Four infected snails were detected in one of these areas, equivalent to a frequency of 7.4%. This area is approximately 8 km (5 miles) from the Miami Metro Zoo. The rat lungworm is therefore established not only in Louisiana but also in Florida.

Angiostrongylus cantonensis was also detected in P. maculata in two locations close to New Orleans. Infected snails occurred at a frequency of about 13% (8/60) in Gretna, Louisiana and 2.5% (1/40) in Mandeville, Louisiana. These two P. maculata populations are in separate drainage basins, in principally urban areas that are close to metropolitan New Orleans, 10 and 50 km (6 and 30 miles) away, respectively. A third sample of P. maculata taken from Schriever, Louisiana, tested negative. The Schriever population is in a drainage basin hydrologically distinct from both the Gretna and Mandeville populations, in a comparatively less urban area.

Although A. cantonensis is established in some parts of Louisiana, it is not known to what extent it has become established in Texas and Mississippi, the two Gulf states adjacent to Louisiana. Infection of a horse (accidental host) by A. cantonensis was reported in Picayune, Mississippi, in 1998.⁹ In 2008, P. maculata became established in a residential lake in Picayune, suggesting that it could serve as a new reservoir for the parasite at this location. However, no Pomacea maculata from this lake tested positive for A. cantonensis. Pomacea maculata from the Houston, Texas, area also tested negative.

Discussion

This study has shown that Pomacea maculata populations infected with A. cantonensis are currently limited to Louisiana within the southeastern United States (Table 1, Figure 1). Detection of A. cantonensis in Gretna and Mandeville confirms the establishment of the parasite in the region and indicates that P. maculata has become a component of the mollusk reservoir for the parasite. A recent survey in China suggests that invasive apple snails (in this case Pomacea canaliculata) can facilitate the spread of A. cantonensis by providing an abundant mollusk reservoir to facilitate parasite reproduction.¹⁵ The present study suggests that nonindigenous apple snails could similarly facilitate the spread of A. cantonensis in other regions and states in the southeastern United States as P. maculata further expands its range.

Although the present survey was limited in scope, there was no evidence to suggest widespread infection of P. maculata in states other than Louisiana. All samples tested in Texas, Mississippi, and Florida tested negative for the parasite. This may reflect an actual absence of A. cantonensis in these locations or the small number of sample locations tested. Despite A. cantonensis having been previously detected in a horse in Picayune, Mississippi,⁹ it was not detected in P. maculata from this location. However, a similar finding occurred in Yunnan, China, where a recent survey revealed no infections of apple snails (in this case P. canaliculata) despite a previous history of A. cantonensis in the region.¹⁵

The negative results obtained with apple snails from Miami should also be considered tentative in view of other data suggesting A. cantonensis is established in the existing rat and terrestrial mollusk populations there. In addition to the infected gibbon at the Miami Metro Zoo, a black rat infected with A. cantonensis was detected in 2004 (Christine Miller, Miami Metro Zoo, oral communication to John Teem, February 2010), supporting the notion that A. cantonensis was present in the area at the time of the gibbon infection. Although it seems unlikely that A. cantonensis could be eliminated from a region once established, apple snail infection rates may fluctuate in response to unknown parameters such as the densities of local rat and mollusk hosts. Rat lungworm may therefore have been present in Miami prior to the appearance of the giant African land snail, but it may also have been brought in with this species at the time of its introduction.

The age of the P. maculata population may also be related to the level of infection and hence likelihood of detecting A. cantonensis in an area where it had previously been detected. For example, the P. maculata population in Schriever (in a drainage basin adjacent to the watershed in which New Orleans is located and where A. cantonensis has been present for at least 20 years), tested negative for A. cantonensis. However, this population has only been established since 2006, and the lack of infection may reflect a time lag for the newly established nonindigenous species to be incorporated into the pool of infected endemic mollusks.

The incidence of P. maculata infection in Louisiana was 6.4% (9/140), similar to the 6.8% for P. canaliculata in China.¹⁵ However, it seems unlikely that the risk of human infection in Louisiana is similar to that in China, where the raw snails are considered a delicacy. Human infections of A. cantonensis in Louisiana have been attributed to eating uncooked infected mollusks or paratenic hosts,^16,17 but infections are rare because such behavior is uncommon. There is, however, a level of potential risk that cannot at present be quantified, but that will continue to exist since these snails are serving as a reservoir for the disease.

Another pathway for human infection by A. cantonensis has been described recently in Hawai‘i. Although Hawai‘i has numerous nonindigenous snail species shown to be infected with A. cantonensis, including P. canaliculata and A. fulica (JR Kim, oral presentation, Rat Lungworm Disease Scientific Workshop, Honolulu, Hawai‘i, August 2011), most of the recent cases of human infection were thought to be associated not with these species but with a recently introduced nonindigenous semi-slug, Parmarion martensi.¹⁸ Human infections appear to result from accidental ingestion of small infected slugs or parts of slugs that are present on raw produce consumed from home gardens. Parmarion martensi is only one of many mollusk species in Hawai‘i that can harbor A. cantonensis; however, the tendency of this species to associate with garden produce that is consumed uncooked may be an important factor in its role in human disease transmission. Parmarion martensi has not been detected in the southeastern United States but mollusk species (native or nonindigenous) with ecological traits and behavior like that of P. martensi may become infected with A. cantonensis and similarly increase the potential for human infections in the region. Apple snails and giant African land snails may thus contribute indirectly to human infections by providing a reservoir for A. cantonensis to infect other mollusk species in the same area that directly infect humans by their association with uncooked foods.

The detection of A. cantonensis in P. maculata in Louisiana and in A. fulica in Florida indicates that nonindigenous mollusks are presently serving as reservoirs to allow A. cantonensis to expand its range to other Gulf of Mexico states. Further research to define the human health risks associated with A. cantonensis as a result of nonindigenous mollusk introductions in the region should therefore include a regional survey of infection rates for both native and nonindigenous mollusk species in the southeastern United States.

Conflict of Interest

None of the authors identifies any conflict of interest.