Release of Angiostrongylus cantonensis larvae

from live intermediate hosts under stress stimuli

Anna Šipková
Masaryk University
Lucia Anettová
Masaryk University
Elena Izquierdo-Rodriguez
University of La Laguna
Vivienne Velič
University of Veterinary Sciences Brno
David Modrý

Masaryk University

Research Article

Keywords: Angiostrongylus cantonensis, stress stimuli, larvae release, gastropods

Posted Date: February 27th, 2024

DOI: https://doi.org/10.21203/rs.3.rs-3973722/v1

License:   This work is licensed under a Creative Commons Attribution 4.0 International License.
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Additional Declarations: No competing interests reported.

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Abstract
The metastrongyloid nematode Angiostrongylus cantonensis is known to cause eosinophilic meningitis
in a variety of homeothermic hosts including humans. Third-stage infectious larvae develop in
gastropods as intermediate hosts. Humans are usually infected by intentional or incidental ingestion of
an infected mollusc or paratenic host (poikilotherm vertebrates and invertebrates). The infection may
also hypothetically occur through ingestion of food or water contaminated by third-stage larvae
spontaneously released from gastropods. Larvae are thought to be released in greater numbers from the
intermediate host exposed to stress stimuli. This study aimed to compare larval release from gastropods
with and without stress stimuli. Experimentally infected Limax maximus and Lissachatina fulica were
exposed to a stress stimulus (shaking on an orbital shaker). The mucus was collected before and after
the stress and examined microscopically and by qPCR for the presence of A. cantonensis larvae and the
DNA. In the case of L. maximus, no larvae were detected microscopically in the mucus, but qPCR analysis
confirmed the presence of A. cantonensis DNA in all experimental replicates, without clear differences
between stress and non-stress period. In contrast, individual larvae of A. cantonensis were found in
mucus from Li. fulica after stress exposure which corresponds to an increased number of DNA-positive
mucus samples after stress. Apparently, stress stimuli of intensity comparable to transport or snail
handling stimulate larval release, especially from highly infected intermediate hosts. However, the small
number of larvae released probably does not pose a significant risk of human infection.

Introduction
Angiostrongylus cantonensis is an invasive parasitic metastrongyloid nematode (Chen 1935; Cowie
2013a). Its heteroxenous life cycle involves rodents, especially Rattus spp., as a definitive host. First-
stage larvae are released in feces and ingested by gastropods (= intermediate hosts), where develop into
third-stage larvae infectious for the definitive host (Cowie 2013b). Third-stage larvae can further survive
in paratenic hosts, such as frogs or lizards (Anettová et al. 2022; Ash 1968; Rosen 1962). This complex
cycle can lead to accidental infections of avian or mammalian hosts by various infection pathways. In
these aberrant hosts, however, the parasite causes severe neurological disease, known as
Angiostrongylus eosinophilic meningitis (AEM) or ocular infections (Diao et al. 2011; Duffy et al. 2004;
Lunn et al. 2012; Monks et al. 2005; Wang et al. 2008; Wright et al. 1991).

In humans, a typical way to acquire infection is by ingestion of raw or undercooked intermediate hosts
(gastropods), transient hosts (crustaceans, centipedes), and paratenic hosts (reptiles, amphibians) that
contain third-stage larvae (L3) (Turck et al. 2022; Pai et al. 2013; Anettová et al. 2022; Federspiel et al.
2020). Consumption of vegetables or water contaminated by L3 released from intermediate hosts is
another way of infection (Chen and Alicata 1964; Cowie 2013b). While liberation of L3 from dead (e.g.
drowned) gastropods is well documented (Modrý et al. 2021; Howe et al. 2019), spontaneous emergence
of infective larvae from live hosts and factors impacting this phenomenon remains a most neglected part
of A. cantonensis transmission cycles (Heyneman and Lim 1967). Recently, Rollins et al. (2023)

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demonstrated an increased number of L3 emerging from Parmarion martensi in response to elevated
temperature, molluscicide, and mechanical disturbance.

Part of human cases diagnosed with AEM is causally connected to ingestion of raw intermediate or
paratenic hosts; however, many cases remain without a confirmed infection source (Wang et al. 2008;
Federspiel et al. 2020; Pandian et al. 2023). The clinical cases in children in endemic area in Mayotte
were tentatively associated with the handling of presumably infected giant African snails Lissachatina
fulica (Epelboin et al. 2016).

Li. fulica is an invasive gastropod species that successfully colonized tropical regions around the world
(Silva et al. 2022; Ayyagari and Sreerama 2017). Due to its size, attractive appearance, and abundance of
peridomestic space, this snail species is commonly handled by humans. We hypothesize that
manipulation or unwanted transporting of molluscs represents a stress stimulus that can increase the
release of L3 and increase the chance of accidental infection in humans. This study on artificially
infected stressed giant African snails and European slugs further corroborates on active emergence of A.
cantonensis infective larvae from their molluscan hosts.

Materials and methods

Animals
The experimental field isolate of A. cantonensis was brought from Fatu Hiva, French Polynesia in 2017.
The life cycle was maintained in laboratory conditions (Modrý et al. 2021) using laboratory rats (Rattus
norvegicus, Wistar strain) and experimental laboratory-reared gastropods: Lissachatina fulica. The
identity of the isolate was confirmed based on the morphology of adult nematodes obtained from
infected rats and by cox1 sequencing, with the haplotype identified as part of the A. cantonensis clade 2
(Červená et al. 2019). Two species of experimental gastropods were selected. Li. fulica is an invasive
species commonly associated with human infections by A. cantonensis; Limax maximus is a broadly
distributed European gastropod proven as a suitable intermediate host of A. cantonensis in temperate
Europe (Anettová et al. 2023; Lima et al. 2020). Captive subadult Li. fulica (Fig. 1a) was purchased from
a private breeder, and the great grey slugs L. maximus (Fig. 1b) were collected from the garden near Brno
in autumn 2021. The molluscs were divided into plastic boxes of 10 individuals, provided coconut soil,
shelters, and moss. Approximately one gram of rat feces was added to all boxes each day for 2 weeks.
Baerman’s method was used to determine the number of larvae per gram of rat feces. As a result,
molluscs of each batch received approximately 40 000 first-stage larvae within two weeks. After the
infection period, molluscs were divided into 7 experimental groups and left for 4 weeks to develop L3.

In total, 84 adult L. maximus slugs of an average weight of 2.2g (± 0.6) were divided into 3 experimental
groups (M1–22 individuals, M2–26 individuals, M3–30 individuals) and a positive control group of 6
individuals; different slug numbers in groups are caused by mortality during the L3 development period.
Similarly, African giant snails Li. fulica (31 in total) with an average weight of 37.3g (± 1.8) were divided

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into 2 experimental groups (G1–12 individuals; G2–12 individuals) and a positive control group of 7
individuals.

The emergence of A. cantonensis third-stage larvae

All gastropods were first rinsed with tap water to remove the residual substrate and all individuals of the
given group were placed in a separate plastic box with perforated lid. In the first experiment, the molluscs
were left in boxes without any stress stimuli for 4 hours at 22°C, exposed to daylight. After this period,
individuals of L. maximus were placed in 50 ml falcon tubes with 15 ml of tap water and gently shaken
for 30 seconds to collect the mucus produced. Considering their larger size, Li. fulica individuals were
placed in a funnel and washed with 15 ml of water, which was collected directly into 50 ml falcon tubes.
The mucus from the boxes was thoroughly shaken with 50 ml of water and collected. The samples were
centrifuged (1 min, 1500 g) and stored for subsequent DNA extraction and qPCR analysis. In the second
experiment, an orbital shaker was used as a stress stimulus. Each box with experimental molluscs was
fixed on a shaker with adhesive tape and shaken for 4 hours at 130 rpm (Fig. 1c). The produced mucus
from individuals and boxes was washed out in a similar way as described above. Positive controls were
left untreated and served for confirmation of presence of L3 at the end of the experiment.

Isolation of the DNA from mucus and qPCR analysis

Centrifuged sediment from collected mucus samples was used for the DNA extraction with a DNEasy
BloodandTissue (Qiagen, Germany) extraction kit. The procedure was optimized for L3 of A. cantonensis
when the pre-lyse phase was extended overnight. A species-specific qPCR assay on LightCycler 480 was
used to detect the presence of A. cantonensis DNA in the mucus (Sears et al., 2021); the reagents and
volumes used are described in Table 1. The cycling conditions of the total number of 40 cycles were set
as follows: 95°C for 20 s, 40°C for 1 s, and 60°C for 20 s. DNA from a single L3 isolated by the same
process as the mucus samples, which was 100× diluted, was used as a positive control for qPCR
analysis.; PCR water was used as a negative control. The samples were analysed in duplicates (Ct values
not exceeding 35 were determined as positive).

Tab. 1 Reagents and volumes used for qPCR diagnostics

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Artificial digestion
After the stress experiment, all used molluscs (except group M3) were sacrificed by decapitation and
artificially digested; group M3 group was not sacrificed but subsequently used for other research. Artificial
digestion was used to determine the total number of L3 in individual slugs and snails. The whole bodies
(without shells in the case of Li. fulica) were cut and fully digested in a digestive fluid (3 g pepsin, 28 ml
25% HCl in a total volume of 1 l of distilled water) on a magnetic stirrer set to 600 rpm at 37°C for two
hours. The digested tissue was transferred through a sieve into 50 ml falcon tubes and centrifuged (1
min, 1500 g). The sediment was gradually transferred to Petri dishes and the total number of L3 was
counted under a light microscope (magnification 40×). Positive controls of L. maximus and Li. fulica
were processed by the same method and the presence of L3 in the tissues of infected molluscs was
microscopically confirmed.

Statistics
GraphPad Prism 9.5.1. was used to perform statistical analysis using Fisher's exact test. Two groups
were tested: L. maximus (total number of 78 individuals) and Li. fulica (total number of 24 individuals).
Means, standard deviations, and percentages of positive samples were calculated using Microsoft Excel
to further characterise the data.

Results
All infected individuals of Li. fulica and L. maximus developed L3 larvae of A. cantonensis in their
tissues, which was proven after completion of the stress experiment, when the individuals were sacrificed
and artificially digested. The L3 in tissues were counted and morphologically identified. The average
number of larvae in group M1 was 18 (± 15) larvae per slug, and in group M2 was 18 (± 16), whereas, in

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the case of Li. fulica, the average number of larvae in group G1 was 7354 (± 5428) and in group G2 was
14620 (± 8547). The average number of larvae in the positive control groups was for L. maximus 17 (±
15) and for Li. fulica 11145 (± 7981).

L3 of A. cantonensis were neither detected in the collected mucus from individual L. maximus nor from
boxes contaminated by mucus. However, the presence of A. cantonensis DNA was confirmed by qPCR
analysis. An increase in the number of positive samples after the stress stimulus was observed only in
the group M2, no change was observed in the group M3 while number of positive samples after stress
decreased in the group M1 (Fig. 2). The difference between the number of DNA-positive samples before
and after the stress stimulus was not statistically significant (p = 1.0000). The DNA positivity was
confirmed in the mucus collected from the boxes before and after the stress stimulus in groups M2 and
M3. In group M1 the presence of DNA in the mucus from the box was not confirmed. The Ct values of
mucus samples from boxes and individuals of L. maximus ranged from 13.90 to 34.16.

Out of 24 Li. fulica examined individually, only a single live A. cantonensis larva was detected (individual
from G1, after stress); 4 additional live larvae were captured in the mucus collected from the G2 box after
stress. The difference in the number of DNA-positive samples from individuals before and after stress
stimulus was statistically significant (p = 0.0001). The percentage of positive individual samples before
and after stress stimulus is shown in Fig. 2. The presence of DNA in the mucus from the G1 box was
confirmed before and after the stress stimulus. In the mucus from the G2 box, the presence of DNA was
confirmed only after stress stimulus. The Ct values of mucus samples from boxes and individuals of Li.
fulica ranged from 16.75 to 33.26.

Discussion
Emerging eosinophilic meningitis caused by Angiostrongylus cantonensis is associated with various
transmission pathways (Cowie 2013b). The dominant way of infection of mammalian hosts including
humans is ingestion of raw intermediate host. Ingestion can be intentional as is usual in countries where
raw slugs and snails are consumed as a delicacy (Eamsobhana 2014). Several cases of infection
resulted from intentional eating raw snails or slugs as a bet or dare (Kwon et al. 2013; Thiengo et al.
2010). Transmission can also occur through ingestion of raw paratenic or transient hosts such as
freshwater shrimp, amphibians, or reptiles (Anettová et al. 2022; Lai et al. 2007; Pai et al. 2013). However,
inadvertent ingestion of L3 can also occur with raw vegetable products, where small snail or slug
individuals may be overlooked (Ash 1976; Tsai et al. 2004; Waugh et al., 2005). Finally, humans can get
infected by ingestion of L3 liberated from an intermediate host into drinking water, or by mucus that can
remain on food or hands (Ash 1976; Heyneman and Lim 1967; Howe et al. 2019; Modrý et al. 2021;
Qvarnstrom et al. 2007).

The ability of infective larvae of A. cantonensis to escape from dead intermediate host is notoriously
known and such larvae can survive in aquatic environments for almost a month (Crook et al. 1971; Modrý
et al. 2021). Survival of L3 in drinking water reservoirs has been theorized as a cause of cases of

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eosinophilic meningitis in humans in Hawaii (Howe et al. 2019). A similar release of L3 from dead
experimentally infected Helix aspersa has been described in metastrongyloid nematodes of carnivores,
such as Aelurostrongylus abstrusus and Troglostrongylus brevior (Giannelli et al. 2015). Released L3 can
be further transferred horizontally between individual molluscs in a process described as intermediasis
(Colella et al. 2015; Modrý et al. 2021).

However, L3 can also spontaneously escape from live intermediate hosts, as demonstrated in
Angiostrongylus vasorum, Aelurostrongylus abstrusus, Crenosoma vulpis, Oslerus rostratus, and
Troglostrongylus wilsoni from experimentally infected gastropods B. glabrata and L. maximus (Barçante
et al. 2003; Conboy et al. 2017; Robbins et al. 2021). Reports confirming the spontaneous liberation of A.
cantonensis L3 from live intermediate hosts are sporadic. In some cases, the presence of a low number
of L3 in the mucus was confirmed (Ash 1976; Heyneman and Lim 1967; Kramer et al. 2018; Qvarnstrom
et al. 2007; Waugh et al. 2016), while Campbell and Little (1988) did not confirm the release of L3 from
Limax flavus.

Only two studies dealt with stressors impacting on the larval release. In the case of related species A.
vasorum, Barçante et al. (2003) confirmed L3 shed from Biomphalaria glabrata after exposure to elevated
temperature or light. Recently, Rollins et al. (2023) demonstrated A. cantonensis larvae released from
stressed semi-slugs P. martensi, however, spontaneous liberation of larvae without stress stimuli was not
observed. The design of our research further extends the latter study. Besides detection of parasite DNA
by qPCR, we also investigated the presence of live larvae, using Li. fulica as previously hypothesized
source of human infections, complemented with experimental L. maximus, a European slug species
proven as a sensitive intermediate host. Mechanical shaking was used to mimic mollusc handling or
transport, as infection by larvae liberated this way from infected intermediate hosts was previously
hypothesised (Asato et al. 2004; Wan and Weng 2004).

In the case of L. maximus, our results do not show larvae in the mucus before or after the stress
exposure, supporting the previous negative result of Campbell and Little (1988) with closely related slugs.
However, the presence of A. cantonensis DNA in the collected mucus was confirmed both before and after
the stress stimulus, which may indicate the release of L3 in a very low number. This corresponds to the
overall lower number of L3 found in L. maximus tissues. The mucus produced was not easy to collect, as
it was firmly attached to the surface of both the gastropods and the walls of the boxes, so some material
may have been lost during collection.

On the contrary, we confirmed the presence of live L3 of A. cantonensis in the mucus of Li. fulica,
however, only after the stress stimulus. Also, presence of A. cantonensis DNA in mucus samples showed
a significant increase in number of positive samples in stressed individuals of Li. fulica, which is
consistent with the results of Rollins et al. (2023). However, the numbers of detected larvae were very low
compared to mucus larval load extrapolated from Ct values (qPCR results) in Rollins et al. (2023).
Experiments focused on the release of A. vasorum L3 from aquatic snails also demonstrated higher
numbers of larvae released (Barçante et al. 2003). In the case of experimental Li. fulica, the absolute

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numbers of L3 detected in tissue were much higher than in L. maximus, ranging around 104, comparable
to the highest infection intensities observed in naturally infected Li. fulica (Tesana et al. 2009). The
dependence of higher larval excretion on infection intensity in intermediate hosts was confirmed in a
study by Rollins et al. (2023) and this may have been a key factor for the successful release of the L3 in
our experiments.

Based on discussed results, we assume that stress stimuli of intensity comparable to mollusc
transportation or handling could elicit larval release from heavily infected individuals. The invasive giant
African snail Li. fulica is common in peridomestic environments both in rural and urban areas and
frequent contact with these snails in A. cantonensis endemic areas was mentioned as one of the possible
risk factors (Epelboin et al. 2016). However, our data suggests that handling or transport of molluscs
infected by A. cantonensis probably does not pose a significant risk of human infection due to the small
number of larvae released even from heavily infected hosts.

Declarations
Author Contribution: Inventing and designing, D.M., and A.Š.; methodology, D.M. and A.Š.; data collection,
A.Š., L.A., E.I.R., and V.V.; project administration, D.M; supervision, D.M; drafting the manuscript, A.Š., and
D.M. and all authors contributed to the final manuscript.

Financial support: The study was supported by Czech Science Foundation grant no. 22-26136S; work of
Anna Šipková and Lucia Anettová was supported by the project MUNI/A/1488/2021 and work of Elena
Izquierdo-Rodriguez was supported by Becas M-ULL (convocatoria 2019).

Data availability: All data obtained in this study are included in the article.

Ethical Standards: The authors declare that all ethical standards were fulfilled.

Consent to participate: The authors consent to participate in this study.

Consent to publication: The authors consent to publish the manuscript.

Competing interests: The authors declare there are no conflicts of interest.

Acknowledgements: The authors acknowledge the Department of Pathology and Parasitology of Vetuni
Brno for providing experimental facilities.

References
1. Anettová L, Izquierdo-Rodriguez E, Foronda P, Baláž V, Novotný L, Modrý D (2022) Endemic lizard
Gallotia galloti is a paratenic host of invasive Angiostrongylus cantonensis in Tenerife, Spain.
Parasitology 149(7), 934–939. https://doi.org/10.1017/S0031182022000336

Page 8/14
 2. Asato R, Taira K, Nakamura M, Kudaka J, Itokazu K, Kawanaka M (2004) Changing epidemiology of
Angiostrongyliasis cantonensis in Okinawa prefecture, Japan. Jpn. J. Infect. Dis 57
3. Ash LR (1968) The occurrence of Angiostrongylus cantonensis in frogs of New Caledonia with
observations on paratenic hosts of Metastrongyles. The Journal of Parasitology 54(3)
4. Ash LR (1976) Observations on the role of mollusks and planarians in the transmission of
Angiostrongylus cantonensis infection to man in New Caledonia. Rev. Biol. Trop 24(1)
5. Ayyagari VS, Sreerama K (2017) Evaluation of haplotype diversity of Achatina fulica (Lissachatina)
[Bowdich] from Indian sub-continent by means of 16S rDNA sequence and its phylogenetic
relationships with other global populations. 3 Biotech 7(4), 252. https://doi.org/10.1007/s13205-
017-0877-4
6. Barçante TA, Barçante JMP, Dias SRC, Lima WS (2003) Angiostrongylus vasorum (Baillet, 1866)
Kamensky, 1905: Emergence of third-stage larvae from infected Biomphalaria glabrata snails.
Parasitology Research 91(6), 471–475. https://doi.org/10.1007/s00436-003-1000-9
7. Campbell BG, Little MD (1988) The finding of Angiostrongylus cantonensis in rats in New Orleans.
The American journal of tropical medicine and hygiene 38(3), 568-73.
https://doi.org/10.4269/ajtmh.1988.38.568
8. Červená B, Modrý D, Fecková B, Hrazdilová K, Foronda P, Alonso AM, Lee R, Walker J, Niebuhr CN,
Malik R, Šlapeta, J (2019) Low diversity of Angiostrongylus cantonensis complete mitochondrial
DNA sequences from Australia, Hawaii, French Polynesia and the Canary Islands revealed using
whole genome next-generation sequencing. Parasites and Vectors 12(1).
https://doi.org/10.1186/s13071-019-3491-y
9. Chen HT (1935) Un nouveau nematode pulmonaire, Pulmonema cantonensis, n. g., n. sp. Annales de
parasitologie humaine et comparée 13(4), 312–317. https://doi.org/10.1051/parasite/1935134312
10. Chen TC, Alicata JE (1964) Possible role of water in the transmission of Angiostrongylus
cantonensis (Nematoda: Metastrongylidae). Journal of Parasitology 50(3), 39–40
11. Colella V, Giannelli A, Brianti E, Ramos RAN, Cantacessi C, Dantas-Torres F, Otranto D (2015) Feline
lungworms unlock a novel mode of parasite transmission. Scientific Reports 5.
https://doi.org/10.1038/srep13105
12. Conboy G, Guselle N, Schaper R (2017) Spontaneous shedding of Metastrongyloid third-stage larvae
by experimentally infected Limax maximus. Parasitology Research 116, 41–54.
https://doi.org/10.1007/s00436-017-5490-2
13. Cowie RH (2013a) Biology, systematics, life cycle, and distribution of Angiostrongylus cantonensis,
the cause of rat lungworm disease. Hawaii Journal of Medicine and Public Health 72(6), 6-9
14. Cowie RH (2013b) Pathways for transmission of angiostrongyliasis and the risk of disease
associated with them. Hawaii Journal of Medicine and Public Health 72(6), 70-4
15. Crook JR, Fulton SE, Supanwong K (1971) The infectivity of third stage Angiostrongylus cantonensis
larvae shed from drowned Achatina fulica snails and the effect of chemical agents on infectivity.

Page 9/14
 Transactions of the Royal Society of Tropical Medicine and Hygiene 65(5), 602–605.
https://doi.org/10.1016/0035-9203(71)90043-5
16. Diao Z, Wang J, Qi H, Li X, Zheng X, Yin C (2011) Human ocular angiostrongyliasis: A literature
review. Tropical Doctor 41(2), 76–78. https://doi.org/10.1258/td.2010.100294
17. Duffy MS, Miller CL, Kinsella JM, de Lahunta A (2004) Parastrongylus cantonensis in a nonhuman
primate, Florida. Emerging Infectious Diseases 10(12)
18. Eamsobhana P (2014) Review paper eosinophilic meningitis caused by Angiostrongylus cantonensis
- a neglected disease with escalating importance. Tropical Biomedicine 31(4)
19. Epelboin L, Blondé R, Chamouine A, Chrisment A, Diancourt L, Villemant N, Atale A, Cadix C, Caro V,
Malvy D, Collet L (2016) Angiostrongylus cantonensis infection on Mayotte Island, Indian Ocean,
2007-2012. PLoS Neglected Tropical Diseases 10(5). https://doi.org/10.1371/journal.pntd.0004635
20. Federspiel F, Skovmand S, Skarphedinsson S (2020) Eosinophilic meningitis due to Angiostrongylus
cantonensis in Europe. International journal of infectious diseases, 93, 28–39.
https://doi.org/10.1016/j.ijid.2020.01.012
21. Giannelli A, Colella V, Abramo F, do Nascimento Ramos RA, Falsone L, Brianti E, Varcasia A, Dantas-
Torres F, Knaus M, Fox MT, Otranto D (2015) Release of lungworm larvae from snails in the
environment: Potential for alternative transmission pathways. PLoS Neglected Tropical Diseases
9(4), e0003722. https://doi.org/10.1371/journal.pntd.0003722
22. Heyneman D, Lim BL (1967) Angiostrongylus cantonensis: Proof of direct transmission with its
epidemiological implications. Science 158(3804), 1057–1058.
https://doi.org/10.1126/science.158.3804.1057
23. Howe K, Kaluna L, Lozano A, Fischer BT, Tagami Y, McHugh R, Jarvi S (2019) Water transmission
potential of Angiostrongylus cantonensis: Larval viability and effectiveness of rainwater catchment
sediment filters. PLoS ONE 14(4). https://doi.org/10.1371/journal.pone.0209813
24. Kramer KJ, Posner J, Gosnell WL (2018) Role of Gastropod Mucus in the transmission of
Angiostrongylus cantonensis, a potentially serious neurological infection. ACS Chemical
Neuroscience 9(4), 629–632. https://doi.org/10.1021/acschemneuro.7b00491
25. Kwon E, Ferguson TM, Park SY, Manuzak A, Qvarnstrom Y, Stephen M, Ciminera P, Murphy GS (2013)
A severe case of Angiostrongylus eosinophilic meningitis with encephalitis and neurologic sequelae
in Hawai’i. Hawaii Journal of Medicine and Public Health 72(6), 41-5
26. Lai CH, Yen CM, Chin C, Chung HC, Kuo HC, Lin HH (2007) Eosinophilic meningitis caused by
Angiostrongylus cantonensis after ingestion of raw frogs. The American Journal of Tropical
Medicine and Hygiene 76(2), 399–402
27. Lima MG, Augusto RC, Pinheiro J, Thiengo SC (2020) Physiology and immunity of the invasive giant
African snail, Achatina (Lissachatina) fulica, intermediate host of Angiostrongylus cantonensis.
Developmental and comparative immunology, 105, 103579.
https://doi.org/10.1016/j.dci.2019.103579

Page 10/14
28. Lunn JA, Lee R, Smaller J, MacKay BM, King T, Hunt GB, Martin P, Krockenberger MB, Spielman D,
Malik R (2012) Twenty two cases of canine neural angiostronglyosis in eastern Australia (2002-
2005) and a review of the literature. Parasites and Vectors 5(1). https://doi.org/10.1186/1756-3305-
5-70
29. Modrý D, Fecková B, Putnová B, Manalo SM, Otranto D (2021) Alternative pathways in
Angiostrongylus cantonensis (Metastrongyloidea: Angiostrongylidae) transmission. Parasitology
148(2), 167–173. https://doi.org/10.1017/S0031182020001857
30. Monks DJ, Carlisle MS, Carrigan M, Rose K, Spratt D, Gallagher A, Prociv P (2005) Angiostrongylus
cantonensis as a cause of cerebrospinal disease in a yellow-tailed black cockatoo (Calyptorhynchus
funereus) and two tawny frogmouths (Podargus strigoides). Journal of Avian Medicine and Surgery
19(4), 289–293. https://doi.org/10.1647/2004-024.1
31. Pai S, Madi D, Achappa B, Mahalingam S, Kendambadi R (2013) An interesting case of eosinophilic
meningitis. Journal of Clinical and Diagnostic Research 7(4), 734–735.
https://doi.org/10.7860/JCDR/2013/4743.2897
32. Pandian D, Najer T, Modrý D (2023) An Overview of Angiostrongylus cantonensis (Nematoda:
Angiostrongylidae), an Emerging cause of human angiostrongylosis on the Indian subcontinent.
Pathogens 12(6), 851. https://doi.org/10.3390/pathogens12060851
33. Qvarnstrom Y, Sullivan JJ, Bishop HS, Hollingsworth R, Da Silva AJ (2007) PCR-based detection of
Angiostrongylus cantonensis in tissue and mucus secretions from molluscan hosts. Applied and
Environmental Microbiology 73(5), 1415–1419. https://doi.org/10.1128/AEM.01968-06
34. Robbins W, Conboy G, Greenwood S, Schaper R (2021) Infectivity of gastropod-shed third-stage
larvae of Angiostrongylus vasorum and Crenosoma vulpis to dogs. Parasites and Vectors 14(1).
https://doi.org/10.1186/s13071-021-04802-6
35. Rollins RL, Medeiros MCI, Cowie RH (2023) Stressed snails release Angiostrongylus cantonensis (rat
lungworm) larvae in their slime. One health 17, 100658.
https://doi.org/10.1016/j.onehlt.2023.100658
36. Rosen L, Chappell R, Laqueur GL, Wallace GD, Weinstein PP (1962) Eosinophilic meningoencephalitis
caused by a metastrongylid lung-worm of rats. JAMA 179(8), 620.
https://doi.org/10.1001/jama.1962.03050080032007
37. Sears WJ, Qvarnstrom Y, Dahlstrom E, Snook K, Kalun L, Baláž V, Feckova B, Šlapeta J, Modry D,
Jarvi S, Nutman TB (2021) AcanR3990 qPCR: A novel, highly sensitive, bioinformatically-informed
assay to detect Angiostrongylus cantonensis infections. Clinical Infectious Diseases 73(7), E1594–
E1600. https://doi.org/10.1093/cid/ciaa1791
38. Silva GM, Thiengo SC, Sierpe Jeraldo VL, Rego MIF, Silva ABP, Rodrigues PS, Gomes SR (2022) The
invasive giant African land snail, Achatina fulica (Gastropoda: Pulmonata): global geographical
distribution of this species as host of nematodes of medical and veterinary importance. Journal of
helminthology 96, e86. https://doi.org/10.1017/S0022149X22000761

Page 11/14
39. Tesana S, Srisawangwong T, Sithithaworn P, Laha T, Andrews R (2009) Prevalence and intensity of
infection with third stage larvae of Angiostrongylus cantonensis in mollusks from Northeast
Thailand. The American Journal of Tropical Medicine and Hygiene 80(6), 983–987
40. Thiengo SC, Maldonado A, Mota EM, Torres EJL, Caldeira R, Carvalho OS, Oliveira APM, Simões RO,
Fernandez MA, Lanfredi RM (2010) The giant African snail Achatina fulica as natural intermediate
host of Angiostrongylus cantonensis in Pernambuco, northeast Brazil. Acta Tropica 115(3), 194–
199. https://doi.org/10.1016/j.actatropica.2010.01.005
41. Tsai HC, Lee SS, Huang CK, Yen CM, Chen ER, Liu YC (2004). Outbreak of eosinophilic meningitis
associated with drinking raw vegetable juice in southern Taiwan. The American journal of tropical
medicine and hygiene, 71(2), 222–226
42. Turck HC, Fox MT, Cowie RH (2022) Paratenic hosts of Angiostrongylus cantonensis and their
relation to human neuroangiostrongyliasis globally. One Health 15, 100426.
https://doi.org/10.1016/j.onehlt.2022.100426
43. Wan KS, Weng WC (2004) Eosinophilic meningitis in a child raising snails as pets. Acta Tropica
90(1), 51–53. https://doi.org/10.1016/j.actatropica.2003.09.019
44. Wang QP, Lai DH, Zhu XQ, Chen XG, Lun ZR (2008) Human angiostrongyliasis. The Lancet. Infectious
Diseases 8(10), 621-30. https://doi.org/10.1016/S1473-3099(08)70229-9
45. Waugh CA, Lindo JF, Lorenzo-Morales J, Robinson RD (2016) An epidemiological study of A.
cantonensis in Jamaica subsequent to an outbreak of human cases of eosinophilic meningitis in
2000. Parasitology 143(9), 1211–1217. https://doi.org/10.1017/S0031182016000640
46. Waugh CA, Shafir S, Wise M, Robinson RD, Eberhard ML, Lindo JF (2005) Human Angiostrongylus
cantonensis, Jamaica. Emerging Infectious Diseases 11(12).
https://doi.org/10.3201/eid1112.050217
47. Wright J, Kelly W, Hamilton J, Wadell A (1991) Equine neural angiostrongylosis. Australian Veterinary
Journal 68(2), 58–60. https://doi.org/10.1111/j.1751-0813.1991.tb03131.x

Figures

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Figure 1

(a) Lissachatina fulica in experimental box during the non-stress phase of the experiment (in the stress
period, snails were partly retracted into the shell); (b) Limax maximus during the non-stress phase of the
experiment; (c) Experimental design of the stress stimulation: box with experimental Li. fulica attached to
the orbital shaker

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Figure 2

Percentage of samples positive for DNA of A. cantonensis by qPCR in the experiment with Limax
maximus and Lissachatina fulica before and after stress stimulus (DNA extracted from mucus collected
from each individual); M1, M2, M3 = experimental groups of L. maximus; G1, G2 = experimental groups of
Li. fulica

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