Journal of Aquatic Animal Health 31:168–172, 2019
© 2019 American Fisheries Society
ISSN: 0899-7659 print / 1548-8667 online
DOI: 10.1002/aah.10063
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Frequency of Occurrence of the Rat Lungworm Parasite in the Invasive
Island Apple Snail in South Carolina, USA
Elizabeth B. Underwood,* Matt J. Walker, Tanya L. Darden, and Peter R. Kingsley-Smith
South Carolina Department of Natural Resources, Marine Resources Research Institute, 217 Fort Johnson Road,
Charleston, South Carolina 29412, USA
Abstract
The rat lungworm Angiostrongylus cantonensis is a nematode
parasite that can cause potentially fatal eosinophilic meningitis in
humans. The life cycle of A. cantonensis involves multiple hosts,
with the most common terminal hosts being rodents and intermedi-
ate hosts comprising gastropods. One such gastropod is the inva-
sive island apple snail Pomacea maculata, which is native to South
America but is currently established in several states in the USA,
including South Carolina. It has been identified as an intermediate
host for A. cantonensis in several locations in Louisiana. The abil-
ity of the island apple snail to serve as an intermediate host for A.
cantonensis poses significant potential threats to human health, yet
no studies to date have determined the prevalence of this parasite
in island apple snails in South Carolina. The objective of this study
was to investigate the frequency of occurrence of A. cantonensis in
South Carolina island apple snails by using a real-time PCR
assay. One-hundred individuals from each of three distinct
stormwater retention ponds were tested, and no positive detections
were found. Determining the prevalence of A. cantonensis in island
apple snails is critical in accurately informing the public as to the
risks involved in handling and/or consuming island apple snails.
The rat lungworm Angiostrongylus cantonensis (Nema-
toda: Metastrongylidae) is a parasitic nematode that is
currently established in southeast Asia, Australia, the
Pacific Islands, the Caribbean, South America, and North
America (Kliks and Palumbo 1992; Wang et al. 2008;
Dorta-Contreras et al. 2011). Angiostrongylus cantonensis
is thought to have been introduced into the USA during
the 1980s from infected rats traveling on ships arriving in
New Orleans (Campbell and Little 1988). The life cycle of
A. cantonensis involves multiple host species. The terminal
hosts for A. cantonensis are several species of rodents, typ-
ically in the genus Rattus, while intermediate hosts are
*Corresponding author: underwoode@dnr.sc.gov
Received November 15, 2018; accepted January 20, 2019
168
predominantly gastropods (Wang et al. 2012; Cowie
2013). Angiostrongylus cantonensis adults reside in the pul-
monary arteries of rats, where mature female parasites lay
eggs, and the resulting larvae (L1) are coughed up, swal-
lowed, and then shed in feces. Gastropods ingest these L1
parasites, which develop into infective third-stage larvae
(L3) within the gastropods, and the gastropods are then
ingested by rats (Wang et al. 2012; Cowie 2013). The L3-
stage nematodes travel in the bloodstream to the central
nervous system of the rats, develop into immature adults,
and finally move to the pulmonary artery, where they
mature, and the cycle starts over again.
In the USA, several invasive gastropod species have
been identified as intermediate hosts of A. cantonensis,
including the golden apple snail Pomacea canaliculata
(Gastropoda: Ampullariidae), the giant African land snail
Achatina fulica (Gastropoda: Achatinidae), and the island
apple snail Pomacea maculata (Gastropoda: Ampullari-
idae), which is synonymous with Pomacea insularum
(Qvarnstrom et al. 2010; Teem et al. 2013; Kim et al.
2014). Studies have shown that such invasive gastropod
hosts can accelerate the spread of A. cantonensis by facili-
tating increased rates of reproduction by the parasite (Lv
et al. 2009a). The ability of these invasive gastropods to
rapidly spread to new areas can subsequently aid in the
spread of A. cantonensis to new regions (Lv et al. 2009a).
Positive detections of A. cantonensis in island apple snails
specifically have been confirmed for three cities in Louisi-
ana: Gretna (frequency of 13.3%; Teem et al. 2013), Man-
deville (frequency of 2.5%; Teem et al. 2013), and New
Orleans (frequency of 16%; Qvarnstrom et al. 2010).
Incidental hosts of the infective L3-stage nematodes
can include humans (Alicata 1962; Wang et al. 2008),

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non-human primates (Gardiner et al. 1990; Emerson et al.
2013), dogs (Lunn et al. 2012), horses (Costa et al. 2000),
birds (Monks et al. 2005), and Virginia opossums Didel-
phis virginiana (Kim et al. 2002). In these incidental hosts,
the L3s travel to the central nervous system, where their
development is arrested, often causing eosinophilic menin-
gitis, which can be fatal (Kliks and Palumbo 1992; Wang
et al. 2008; Dorta-Contreras et al. 2011). In humans, sev-
eral cases of eosinophilic meningitis caused by A. canto-
nensis have been reported in Louisiana (New et al. 1995),
Texas (Foster et al. 2016), and Tennessee (Flerlage et al.
2017). Angiostrongylus cantonensis has yet to be detected
in island apple snails in South Carolina. This parasite,
however, poses a risk to human health in South Carolina,
as ponds sampled in the state are all located in residential
neighborhoods and are highly accessible, allowing resi-
dents to easily collect island apple snails in shallow water
along the pond banks. Residents in some areas have
reported handling island apple snails—usually out of
curiosity—with bare hands, as they are unaware of the
risks involved with handling these snails without appropri-
ate gear. This method of contact likely poses the greatest
risk of human exposure in South Carolina because island
apple snails are not used as a food source as they are in
other regions. Due to the potential for the island apple
snail to serve as an intermediate host for A. cantonensis
and given its prevalence in suburban areas of the state,
determining the frequency of this parasite in South Caro-
lina island apple snail populations is important from a
human health perspective.
METHODS
To accurately inform the public as to the risks involved
in the handling and/or consumption of island apple snails,
the frequency of occurrence of A. cantonensis in three dis-
crete, established island apple snail populations in South
Carolina was determined using PCR-based detection
assays (Qvarnstrom et al. 2010). Island apple snail individ-
uals were collected from three isolated ponds in South
Carolina (Myrtle Beach, West Ashley, and Mount Pleas-
ant) between 2015 and 2016 (Figure 1; Table 1). After col-
lection, whole snails were placed on ice in a biosecure
container, brought back to the South Carolina Depart-
ment of Natural Resources’ (SCDNR) Marine Resources
Research Institute, and stored in a freezer maintained at

20°C until further processing could be conducted. Dur-
ing the processing of each island apple snail sample
(n = 100 snails for each location; Table 1), a portion of
lung tissue was removed, placed in a vial containing 95%
ethanol, labeled, and stored at
20°C for genetic analyses.
All genetic processing and analyses were conducted at
the Hollings Marine Laboratory in Charleston, South Car-
olina, between June 2016 and June 2017. A modified
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169
FIGURE 1. Sampling locations in Myrtle Beach, Mount Pleasant, and
West Ashley, South Carolina, where island apple snails were collected to
determine the occurrence of rat lungworm Angiostrongylus cantonensis.
Wizard SV Genomic DNA Purification System protocol
(Promega Corporation, Madison, Wisconsin) was used to
isolate DNA from island apple snail lung tissue samples.
Lung tissue was used because Liu et al. (2005) reported
that significantly more A. cantonensis larvae were dis-
tributed in the lung and foot tissues than other tissues in
the congener species, the golden apple snail. Furthermore,
larval density was higher in lung tissue than in other tissues
(Wei et al. 2010). Finally, Lv et al. (2009b) observed A.
cantonensis L1 larvae migrating to the lungs of infected
golden apple snails, where they remained and developed
into L3 larvae. Each tissue sample (0.3–0.5 g) was placed
in 927.3 μL of a digestive solution containing 727.3 μL of
nuclei lysis solution, 181.8 μL of 0.5-M EDTA, and
18.2 μL of water. Samples were then homogenized at
30 Hz using the Qiagen Tissue Lyser for 5 min, after which
time 72.7 μL of 20-mg/mL proteinase K (enzyme code
3.4.21.64; IUBMB 1992) were added, and the samples were
incubated at 55°C for 3 h or until all tissue was digested.
Note that when testing island apple snail samples known
to be infected with A. cantonensis (obtained by Teem et al.
2013 in Louisiana), some samples only amplified when
more tissue was used (compared to Teem et al. 2013 and
Qvarnstrom et al. 2010), the digestion solution was scaled
up, and samples were homogenized. Therefore, these DNA
isolation modifications were used for all samples tested in
the present study in order to detect the parasite in the event
of a low parasite load in individual samples. From each
sample, 200 μL of lysate were then added to 180 μL of
Wizard SV Lysis Buffer and placed in a spin column
assembly. Samples were placed in a centrifuge and spun for
an initial 3 min at 13,000 revolutions per minute (rpm). A

170
TABLE 1. Sampling locations, collection months, sample sizes, and size ranges of island apple snails analyzed from South Carolina ponds to deter-
mine the occurrence of rat lungworm Angiostrongylus cantonensis.
Location Latitude; longitude
Mount Pleasant 32°490 37.21″N; 79°480 40.43″W
Myrtle Beacha 33°390 36.75″N; 79°00 17.60″W
West Ashleya 32°510 1.33″N; 80°50 12.5″W
a
Individuals from the West Ashley and Myrtle Beach samples were collected as part of the research on island apple snails described by Gooding et al. (2018).
series of four total spin cycles (1 min at 13,000 rpm fol-
lowed by the addition of 650 μL of Wizard SV Wash solu-
tion) was conducted. Following a final spin cycle of 3 min
at 13,000 rpm, DNA was eluted with 80 μL of nuclease-
free water at 55°C. Isolated DNA was stored at
20°C
until further analyses were conducted.
The real-time PCR assay was performed in an 11-μL
total reaction volume containing iTaq Universal Probes
Supermix (BioRad, Hercules, California), 0.2 μM of pri-
mers (developed by Qvarnstrom et al. 2010), 0.2 μM of
TaqMan probe, and 1 μL of DNA (1:100 dilution). The
standard cycling conditions for each assay were 94°C for
3 min, followed by 50 cycles of 94°C for 15 s and 60°C
for 1 min. Each PCR assay was run with seven negative
controls to detect any contamination and one positive
control containing DNA isolated from island apple snail
samples known to be infected with A. cantonensis (i.e.,
from snails collected in Louisiana by Teem et al. 2013).
Eight technical replicates were conducted for each individ-
ual screened, and PCR inhibition tests were conducted on
all samples that did not amplify by re-running these sam-
ples with two technical replicates spiked with A. cantonen-
sis control DNA to verify that the negative results were
not false due to chemical inhibition in the reactions. The
frequency of the parasite was calculated by dividing
the number of positive detections for each location by the
total number of individuals screened.
RESULTS
All 300 individual island apple snails from the three
sampling locations combined tested negative for the pres-
ence of A. cantonensis, as none of the eight replicates for
any of the individuals amplified. Furthermore, no chemi-
cal inhibition was detected in any of the reactions.
Although there was a delay of over 1 year in some cases
between the collection of island apple snails and the PCR
assay, it is unlikely that significant degradation of poten-
tial larval DNA occurred in the samples. The total PCR
target fragment is very small—less than 105 base pairs—
so the probability of these fragments staying intact is high,
even if stored at –20°C for over 1 year. In addition, con-
trol tissues known to be infected with the parasite and
control parasite DNA were stored at –20°C for long
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UNDERWOOD ET AL.
Collection month(s) and year Sample size Shell height range (mm)
Jul–Dec 2016 100 27.8–76.5
Oct 2015 100 24.4–74.3
Jun–Oct 2015 100 38.9–89.5
periods of time, and no degradation of this DNA was
observed. Based on our sampling efforts, these results indi-
cate that A. cantonensis was absent from the island apple
snails that were collected in South Carolina.
DISCUSSION
The results of this study indicate that the nematode
parasite A. cantonensis is absent from island apple snail
populations in South Carolina at this time. The number of
samples tested is believed to have been sufficient for
detecting the parasite if it had present in island apple
snails in this region, based upon previously reported mini-
mum frequencies of 2.5% in Louisiana, where the parasite
is established (Teem et al. 2013). Based solely on this
prevalence level, if A. cantonensis is present in island apple
snails in South Carolina, we would predict that 2.5 out of
every 100 individual snails would test positive for the par-
asite, yet our observed value was zero. It remains possible,
however, that A. cantonensis is present in South Carolina
either in other intermediate hosts or in the definitive host
(Rattus spp.), but none of those species was tested as part
of this study. Emerson et al. (2013) tested two native and
one introduced gastropod species in South Carolina using
the real-time PCR assay developed by Qvarnstrom et al.
(2010), and all results were negative for A. cantonensis.
If A. cantonensis is established in South Carolina, it is
important to note that it may not result in the infection of
island apple snail populations. For example, in Picayune,
Louisiana, a horse was infected with A. cantonensis, indi-
cating the occurrence of the parasite in the area, yet no
island apple snail individuals tested were infected with the
parasite (Teem et al. 2013). Furthermore, Lv et al.
(2009a) conducted their study in Yunnan, China, where
A. cantonensis is well established, and they reported a 0%
frequency of the parasite in golden apple snails. The lack
of detections of A. cantonensis in the present study could
also be due to time lags between the establishment of the
parasite and the establishment of island apple snail popu-
lations. It is possible, as suggested by Teem et al. (2013),
that insufficient time has passed for the island apple snails
to become infected. Those authors tested an island apple
snail population that is thought to have become estab-
lished during 2006 in an area where A. cantonensis has

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been established for at least 20 years, yet they did not
detect the parasite in that snail population (Teem et al.
2013). The island apple snail populations sampled in the
present study (Myrtle Beach, Mount Pleasant, and West
Ashley) are believed to have been established in 2008,
2010, and 2013, respectively (Gooding et al. 2018). There-
fore, if A. cantonensis is present in these areas, insufficient
time may have elapsed for the island apple snail popula-
tions to have become infected.
Focusing future research efforts on determining the
prevalence of A. cantonensis in other intermediate hosts or
the definitive host would be informative for an assessment
of potential human health concerns associated with the con-
sumption or handling of such species. If A. cantonensis is
present in South Carolina, in light of the possibility of these
time lags, continued periodic testing of established island
apple snail populations may be warranted. It will also be
important to monitor new populations of island apple snails
for the occurrence of A. cantonensis, if such populations
become established. In summary, the health risks associated
with A. cantonensis warrant the close monitoring of the
potential establishment and spread of this parasite into new
areas in South Carolina. Invasive apple snails have been
shown to act as reservoirs for A. cantonensis, enabling para-
site reproduction and future spread; thus, early detection of
A. cantonensis in apple snail populations could be critical in
limiting the spread of the parasite and the subsequent asso-
ciated health risks (Lv et al. 2009a).
ACKNOWLEDGMENTS
We wish to thank the residents of Village Green, West
Ashley; Ravens Run, Mount Pleasant; and the subdivision
in the Myrtle Beach, South Carolina, sampling area for
allowing researchers to sample the ponds located on pri-
vate property within these areas. We appreciate the
SCDNR Population Genetics Section staff for their gen-
eral assistance in sample processing and the SCDNR
Shellfish Research Section staff for their field assistance.
We are grateful to Isaure De Buron (College of Charles-
ton) for her guidance and expertise throughout this pro-
ject, drawing specifically on her expertise in parasitology.
We also thank Yvonne Qvarnstrom (Centers for Disease
Control and Prevention) and John Teem (Florida Depart-
ment of Agriculture and Consumer Services) for providing
positive A. cantonensis DNA for use in the real-time PCR
assay and for providing guidance at the outset of this pro-
ject. Funding for the project was provided by a grant
awarded to the SCDNR through the U.S. Fish and Wild-
life Service’s State and Interstate Aquatic Nuisance Spe-
cies Management Plan Program. This publication
represents SCDNR Marine Resources Research Institute
Contribution Number 798. There is no conflict of interest
declared in this article.
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171
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