Summary

Gastropod-borne parasitic diseases, such as metastrongyloid lungworm infections of humans and animals, are rarely addressed in research. In Europe, multiple species of metastrongyloid lungworm species infect various definitive hosts, both domestic and free-living animals. Two highly prevalent lungworms, Angiostrongylus vasorum and the closely related Crenosoma striatum are known to infect domestic dogs and certain wildlife. For A. vasorum this includes next to domestic dogs, wild canids (e.g. wolves, foxes, and jackals), badgers, raccoons and red pandas. All these carnivore species act as definitive hosts (DH) where in vivo the adult nematodes reside in the right heart and in the Arteria pulmonalis. Canine angiostrongylosis may lead to a variety of symptoms, ranging from mild to severe ones. Conversely, C. striatum is commonly found in the lungs of European hedgehogs (Erinaceus europaeus). The most common symptoms of crenosomosis in affected hedgehogs impact the respiratory system.

The life cycle of these lungworms is heteroxenous with terrestrial gastropods acting as obligatory intermediate hosts (IH). Terrestrial gastropods become infected by feeding either on the faeces containing first-stage larvae (L1) from an A. vasorum -infected DH or from a C. striatum -infected European hedgehog. While inside the gastropod IH, the larvae develop into the second-stage larvae (L2) and third-stage larvae (L3), which is then infective for other DH.

The giant African snail Lissachatina fulica is one of the largest terrestrial snails in the world. Originally endemic from East Africa, this gastropod species is currently regarded as one of the most invasive gastropod species worldwide. L. fulica is known to be an important IH for multiple parasite species, including various lungworm species.

Gastropod-borne metastrongyloid lungworm infections are poorly understood despite their importance in both veterinary- and human medicine. To obtain precise data on the parasite’s location and distribution speed inside terrestrial gastropods in vivo, imaging techniques, as positron emission tomography and computed tomography (PET/CT) can be used.

This study intends to assess the use of PET/CT to scan radiolabelled A. vasorum – and C. striatum first-stage larvae (L1) while migrating in vivo in the obligate mollusc intermediate hosts. Here, the giant African snail (L. fulica), was used as novel animal model for lungworm-associated investigations as this gastropod species is known to act as natural obligate intermediate host in the tropics for various metastrongyloid lungworms, including the zoonotic-relevant Angiostrongylus cantonesis. ¹⁸F-FDG radiolabelled A. vasorum- and C. striatum L1 migration was visualized through PET/CT imaging after L1 oral infection or injection.

Results from nanoScan® PET/CT

The larvae were retrieved from the fridge in concentrated form, and combined with ¹⁸F-FDG activity at 30 MBq and incubated at RT for 30 min. The larvae were then washed in phosphate-buffered saline (PBS). Finally, the activity was measured and noted down.

Giant African snails (L. fulica) were carefully cleaned with running water before larval infection. The radiolabelled ¹⁸F-FDG L1 suspended in solution were injected near the pneumostome, to inject the larvae suspension close to the gastropod central haemolymph circulation system. After this procedure, the snails were returned inside their boxes to wait for 5 minutes for larval migration. Before PET/CT analysis, the snails were immobilized using an ice bath and placed on the bed for the scanning machine (NanoScan, Mediso).

PET/CT images were acquired in six infected L. fulica at 30-, 60- and 90 min post infectionem (p. i.). A seventh non-infected snail was injected only with ¹⁸F-FDG to serve as control. The CT was obtained using the following parameters: 70 kV, 84μAs, 1 projection. PET scans were performed using a whole-body window. The first scan was a dynamic scan consisting of ten short scans at a one min interval. Images were reconstructed using a reconstruction program using the MLEM algorithm.

PET/CT images revealed hotspots immediately post injection, primarily in the snail's mass dorsally from the pneumostome. These indicated limited dispersal in 30 min. Conversely, 60- and 90-min post injection, the scans using A. vasorum L1 displayed a more widespread larval distribution throughout the gastropod's body indicative of advanced larval migration. The activity, however, was located in the lower digestive tract and the albumen gland. Two snails showed a distinct distribution that was not found in the control ¹⁸F-FDG injected snail, a focal point in the top part of the digestive gland.

The post-injection scans of snails showed larvae activity arising from the injection spot as well as from the lower part of the gastrointestinal tract in four individuals. The remaining scans that were performed every 30 min post-injection demonstrated a wide spread pattern that could not be linked to one specific organ.

Figure 1: PET/CT image of two Lissachatina fulica snails (a, c) and one control (b) scanned 60 minutes after injecting ¹⁸F-FDG radiolabelled Angiostrongylus vasorum larvae. 
a, top; red arrows: activity in digestive tract and digestive gland. b, middle; control. c, bottom; digestive gland visible.

Conclusion

• This study set out to establish whether it was possible to radiolabel metastrongyloid lungworm larvae and track larval migration in vivo within obligate gastropod IH via a PET scan.
• It was feasible to label the parasitic larvae with FDG after 30 min of incubation and that these parasitic stages could be visualized in an early state of their endogenous gastropod migration with a PET/CT scan.
• In this study it was shown that ¹⁸F-FDG radiolabelling of vital L1 of two endemically occurring metastrongyloid species in Europe, i. e. vasorum and C. striatum, can be achieved to track migration routes within gastropod IH with minimal invasive (injection of larvae) and with non-invasive (oral uptake of larvae) methods.
• This initial work should be repeated and tested on a larger sample number of gastropods, with higher infectious doses and with other radiolabelled tracers.
• Thus, different radionuclides as well as other lungworm species, including zoonotic ones (A.  cantonensis, Angiostrongylus costaricensis) can also be considered in the future since L. fulica is known to be a suitable IH in the tropics.
• Detailed in vivo PET/CT scans of gastropods with not only higher infectious doses, other radiolabelled tracers but also longer observation periods might allow detection of either organ tropism or larval accumulation in certain mollusc tissues/organs for further development into infective stages.

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