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 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 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.