Autochthonous Angiostrongylus cantonensis, Angiostrongylus vasorum and Aelurostrongylus abstrusus infections in native terrestrial gastropods from the Macaronesian Archipelago of Spain

The presence of zoonotic relevant Angiostrongylus cantonensis infections has recently been reported in rat final hosts and gastropod intermediate hosts in Tenerife, Spain. However, data on A. cantonensis, Angiostrongylus vasorum and Aelurostrongylus abstrusus prevalences in endemic gastropods for other islands of the Macaronesian Archipelago are still missing. In order to fill this gap, we conducted an epidemiological study on terrestrial native slug (Plutonia lamarckii) and snail (Cornu aspersum, Theba pisana, Rumina decollata) species in 27 selected locations of Tenerife, Gran Canaria, El Hierro, Lanzarote, La Palma and Fuerteventura. Overall, 131 terrestrial gastropods were collected in winter/spring season 2018/2019 and examined for the presence of metastrongyloid lungworm larvae via artificial digestion. The current data revealed a total prevalence of 4.6% for A. vasorum, 3.8% for A. abstrusus and 0.8% for A. cantonensis. In Tenerife, three lungworm species were detected, thereby re-confirming A. cantonensis endemicity for this island. Prevalences of snails (C. aspersum) originating from El Hierro were 5% for A. abstrusus and 15% for A. vasorum, respectively, with larval burdens up to 290 larvae per specimen. This epidemiological study indicates the presence of human, canine and feline lungworm species in Macaronesia, Spain. The current data—particularly those on anthropozoonotic A. cantonensis—call for a regular large-scale monitoring on intermediate hosts, paratenic hosts and definitive hosts to prevent further spread of lungworm-related diseases in humans and animals.


Introduction
Angiostrongylus cantonensis, Angiostrongylus vasorum and Aelurostrongylus abstrusus represent lungworms of the family Metastrongyloidea, which can infect humans, domestic dogs and cats. Recent European surveys indicate that these parasites are spreading within Europe (Foronda et al. 2010;Jefferies et al. 2010a;Schnyder et al. 2017;Penagos-Tabares et al. 2020;Federspiel et al. 2020). The zoonotic parasite A. cantonensis is found in various rat final host species of Australia, China, India, Pakistan, vast areas of Southeast Asia, Pacific and Indian Ocean islands and South America. It is also endemic in Egypt, the Caribbean area and the southern tropical part of the USA. More recently, A. cantonensis has been reported from rats (Rattus rattus) (Foronda et al. 2010) and terrestrial gastropods (Martin-Alonso et al. 2011 of the island Tenerife, Macaronesian Archipelago, and additionally from two hedgehogs of the island Mallorca, Spain (Paredes-Esquivel et al. 2019), proving its geographic expansion into the previously non-endemic European regions (Federspiel et al. 2020). Neurotropic third-stage larvae (L3) of A. cantonensis are the etiological agent of human angiostrongyliasis, which is characterized by severe eosinophilic meningoencephalitis/encephalitis with sometime lethal outcome (Martin-Alonso et al. 2015;Federspiel et al. 2020).
Humans become infected by oral ingestion of raw or undercooked terrestrial gastropods (i.e. slugs/snails) containing infective L3 or by consumption of undercooked paratenic hosts (i.e. amphibians, crabs, crayfishes). In rats, ingested L3 rapidly migrate through intestinal walls, spread haematogenously, reach cerebrum and cerebellum within 4-6 days post infectionem (p. i.), and moult into fourth-stage larvae (L4) which invade the subarachnoid space and migrate via blood circulation to the pulmonary artery and right heart. Conversely, in A. cantonensis-infected humans, neurotropic L4 can reach 1-2 mm in size and are often enclosed by granulomas in parenchyma of cerebrum, cerebellum or in the subarachnoid space, thereby causing severe tissue inflammation (Graeff-Teixeira et al. 2009;Wang et al. 2012; Barratt et al. 2016).
The lungworms A. cantonensis, A. vasorum and A. abstrusus have been considered neglected and underestimated in Europe (Foronda et al. 2010;Martin-Alonso et al. 2011Lange et al. 2018a) and other geographic regions (Traversa et al. 2010;Penagos-Tabares et al. 2018aFederspiel et al. 2020). Nowadays, due to an increased research focus on lungworms, occurrences of these nematodes are reported from several geographical areas of Europe (Morgan and Shaw 2010;Taylor et al. 2015;Barutzki et al. 2017;Maksimov et al. 2017). Since metastrongyloid lungworms require terrestrial gastropods as obligate intermediate hosts to complete their life cycles, knowledge on seasonal infections in gastropods seems crucial for a better understanding of the epidemiology of these parasitoses. Whilst a vast amount of data exists on lungworm infections in definitive hosts (Taubert et al. 2009;Wang et al. 2012;Barutzki and Schaper 2013;Di Cesare et al. 2015;Barratt et al. 2016;Gillis-Germitsch et al. 2017a;Schnyder et al. 2017), little has been reported on natural gastropod infections (Ferdushy et al. 2009;Majoros et al. 2010;Patel et al. 2014;Lange et al. 2018a, b;Penagos-Tabares et al. 2019, 2020Dimzas et al. 2020). To our best knowledge, there is only one report on A. cantonensis-infected slugs/snails in Tenerife (Martin-Alonso et al. 2015).
Therefore, the aim of this study was firstly to evaluate lungworm larval infections in native terrestrial gastropod populations, and secondly, to address a potential geographic expansion of these parasites into other islands of the Macaronesian Archipelago in Spain.

Study area and slug/snail collection
As also true for the Spanish mainland, a temperate Mediterranean climate predominates in the Archipelago of Macaronesia (see Fig. 1). Rainfall can be scarce in distinct islands (i.e. Lanzarote and Fuerteventura), where prolonged dry seasons and hot summers are typical. Each island has an individual microclimate, based on a distinct geography and ecoepidemiology, including endemic vegetation and vertebrate/ invertebrate species diversity. The current terrestrial mollusc collection sites were diverse but semi-arid climate conditions were predominant (see Table 1). Thus, collection sites were mainly composed of xerophyts (e.g. cactus and euphorbia), grassland, shrubs, and also sub-tropical pine and laurel forests (in Tenerife, El Hierro and La Palma) with evergreen endemic trees, such as Pinus canariensis and broadleaf Lauraceae (e.g.
Molluscs were weighted, individually stored in plastics bags, cryo-euthanized according to Lange et al. (2017) and stored frozen at − 20 °C at the Faculty of Veterinary Medicine, University of Las Palmas de Gran Canaria, Spain, until further investigations.
Before artificial digestion, gastropod species were identified based on their morphological characteristics according to literature (Nordsieck 2000;Martin-Alonso et al. 2015). Frozen gastropods were transferred to the Institute of Parasitology at Justus Liebig University Giessen (JLU), Giessen, Germany. According to current national animal protection laws of Spain, a permission for gastropod collection or their use for basic research purposes is not required.

Morphological and morphometric identification of metastrongyloid larvae
Remnants of the last sieving process were transferred into sterile 15-mL Falcon tubes and pelleted at 40 × g for 10 min at room temperature (RT). Pellets were resuspended and examined via an Olympus BH-2® microscope equipped with a digital camera (SC30®, Olympus). Metastrongyloid species and stages were identified

Molecular identification
To confirm morphological findings, DNA from pooled metastrongyloid larvae of single gastropods was isolated using a commercial kit (Quiagen DNeasy Blood and Tissue Kit®) and analysed as described previously (Lange et al. 2018a, b;Penagos-Tabares et al. 2019, 2020. Molecular species confirmation was performed by running conventional PCRs with the universal nematode primers NC1/ NC2 (Gasser et al. 1993) followed by species-specific realtime PCRs. A duplex real-time PCR for A. abstrusus and T. brevior was carried out, amplifying the internal transcribed spacer 2 (ITS-2) region from the ribosomal DNA. This PCR was conducted, using the forward primers TrogloF and AeluroF and the single reverse primer MetR ). The duplex real-time PCR for A. vasorum was performed, amplifying a partial ITS-2 region as reported by Jefferies et al. (2009).
Unfortunately, the samples did not contain sufficient amplifiable DNA for molecular analyses. Therefore, for prevalence calculation, only microscopic-based data was used.
Larval burden per specimen varied considerably from one to 290 larvae for individual gastropods. Overall, 55.6% (5/9) of metastrongyloid-positive gastropods contained less than 10 larvae. Interestingly, the two molluscs (P. lamarckii and C. aspersum) with the highest larval burden (138 and 290 larvae, respectively) both showed a double infection with A. vasorum and A. abstrusus. Coinfections were observed in three gastropods. One gastropod (C. aspersum) contained larvae, which could not be identified due to their destroyed cuticles, oesophagus or tails. Besides lungworm larvae, some typical gastropod-specific parasitic nematodes of the genus Phasmarhabditis were detected, but not further considered in this study.

Discussion and conclusion
The present epidemiological study offers conclusive evidence that native gastropod species in Macaronesia may act as suitable intermediate hosts for human and canine angiostrongylosis and feline aelurostrongylosis, under natural habitat conditions. It further represents the first report in Spain on A. vasorum and A. abstrusus infections, occurring in terrestrial intermediate hosts, thereby underlining their role in the epidemiology of these neglected parasitoses. It also confirms recent reports on the occurrence of anthropozoonotic-relevant A. cantonensis in Tenerife. The biodiversity of terrestrial gastropod species in this particular region of Spain gives evidence that native molluscs will most probably contribute to expansion of zoonotic-and veterinaryrelevant lungworms into other islands where infections of humans, dogs and cats with these lungworms have not yet been reported.
The distribution pattern of metastrongyloid-positive gastropods in Tenerife indicated a widespread geographical extension of these parasites on this island. Whilst other related studies described an occurrence of A. cantonensis in the northeastern part of the island (Foronda et al. 2010;Martin-Alonso et al. 2011, here we detected A. cantonensis infections in the municipality Guia de Isora, which is situated in the western part of Tenerife. A. cantonensis infections remained restricted to this island, thereby confirming its geographic endemicity (Foronda et al. 2010;Martin-Alonso et al. 2011, but denying further expansion into other archipelago islands. Since the Canary Archipelago has a good infrastructure and is popular for tourism, an oversea transportation system by ferries is operated between the islands; thereby, facilitating transmission of lungworm-infected definitive hosts is more likely. Accordingly, we expected to detect more lungworm-positive islands/locations. In Gran Canaria, the second most populated and a well-travelled island of the Canary Archipelago, aelurostrongylosis was diagnosed 2016 for the first time in feral cats (Rodriguez-Ponce et al. 2016). No A. abstrusus-infected gastropods were found in the current study on this island. One explanation for this result may be the limited number of analysed molluscs. The presence of gastropods and their parasite burden seems to be dependent on different climatic, ecological and environmental factors (Lv et al. 2006;Ferdushy et al. 2010;Giannelli et al. 2016). Furthermore, individual and species-specific coprophilic/ coprophagic behaviour in gastropods plays a role in the intermediate host capability to become infected by lungworm larvae. As gastropods are humidity dependent, finding them, in locations with vast semi-arid areas and valleys, as observed in Gran Canaria, is challenging. In the relatively humid island of the Canary Archipelago, La Gomera, which represents an apparently good habitat for gastropods, we expected to find a richer mollusc population. However, on this island, no gastropods were found despite two excursion efforts into different geographic locations.
The current data showed both lungworm coinfections and high parasitic burden in single Macaronesian gastropods. Thus, approximately half of the infected gastropods (5/9) comprised more than one lungworm species. Assuming that a longer exposure to faeces, covering metastrongyloid larvae leads to a higher probability for gastropods to become infected, we also expect that the occurrence of coinfections and the larval burden are dependent on this parameter. Besides age, also coprophagic preferences in oral uptake influence gastropod's exposure to infective L1. Therefore, factors affecting coprophagic behaviour in gastropods should be addressed in future studies. All here investigated metastrongyloid lungworms show a rather broad intermediate host spectrum, implicating that multiple intermediate hosts might exist at current sampling sites. Further studies on complex parasite-intermediate host interactions are currently planned to better understand gastropod-derived innate immune reactions against these larval stages. Consistently, it was recently demonstrated that gastropod-derived haemocytes seem capable to rapidly cast the so-called invertebrate extracellular  (Nogales et al. 2004;Medina and Nogales 2009;Martin-Alonso et al. 2011Rodriguez-Ponce et al. 2016;Carretón et al. 2020), large-scale epidemiological surveys are needed, addressing definitive hosts (dogs, cats, rats) and gastropods as well as paratenic hosts (birds, reptiles, rodents) within this archipelago. Preventive education on this neglected parasitosis as well as food inspections is necessary, as snail consumption by humans is a common practice in Macaronesia. Thus, this work aims to raise awareness of veterinary surgeons, physicians and public health authorities not only in Macaronesia but also in other regions with similar climatic conditions.
Author contribution AR, AT and CH designed the survey. AR and AC collected the samples. LS and AC processed the slugs/snails and carried out microscopy analyses. LS performed the gastropod molecular analyses. LS, AR, AT and CH wrote the manuscript. AT, CH and AR revised the manuscript. All authors read and approved the final manuscript.
Funding Open Access funding enabled and organized by Projekt DEAL. This research was funded by the Institute of Parasitology, Justus Liebig University Giessen, Germany.

Declarations
Ethical statement Not applicable.

Conflict of interest
The authors declare no competing interests.

Animal welfare Not applicable
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