Angiostrongylus vasorum, Aelurostrongylus abstrusus, Crenosoma vulpis and Troglostrongylus brevior Infections in Native Slug Populations of Bavaria and Baden-Wuerttemberg in Germany

Angiostrongylus vasorum, Crenosoma vulpis, Aelurostrongylus abstrusus and Troglostrongylus brevior can cause severe cardiovascular and pulmonary symptoms in companion animals and wildlife. Recently, these nematodes were reported to spread within Europe and South America. The reasons behind this are still unknown, but obligate gastropod intermediate host populations might play a role. Therefore, lungworm infections in terrestrial slug populations in selected geographic areas of the Federal States of Bavaria and of Baden-Wuerttemberg, Germany, were studied. In total, 517 slugs (462 Arion spp., 51 Deroceras reticulatum, one Limax maximus, and three unknown slug species) were collected in the summer and autumn seasons, artificially digested and microscopically and molecularly analyzed for the presence of metastrongyloid lungworm larvae. Overall, gastropods showed a prevalence of 11.61% (60/517) for A. vasorum, 1.74% (9/517) for A. abstrusus, 0.77% (4/517) for C. vulpis and 0.97% (5/517) for T. brevior infections, respectively. In Obrigheim (Baden-Wuerttemberg), a hyperendemic focus of canine angiostrongylosis was identified. Here, gastropod infection rates rose from 13.60% (17/125) to 62.96% (34/54) within a few months. In total, 25.61% (84/328) of analysed terrestrial gastropods from Baden-Wuerttemberg were positive for metastrongyloids. In contrast, Bavarian gastropods showed a much lower prevalence of 4.76% (9/189). For the first time, the presence of T. brevior was confirmed for Arion spp. in Baden-Wuerttemberg via molecular analyses. Overall, the current data confirm that canine angiostrongylosis occurs in hyperendemic foci in certain geographic areas with high infection rates in intermediate host populations. As a result, the prevalence for a specific region can rise remarkably within a short period of time. Thus, for a better understanding of lungworm epidemiology in Germany and to protect dogs from angiostrongylosis in hyperendemic foci, it seems mandatory to enhance current efforts on Metastrongyloidea-targeted monitoring on a geographical and time span-related level.

A. vasorum, also called the French heartworm, is a highly pathogenic lungworm which causes severe and occasionally fatal angiostrongylosis in canine definitive hosts, including

Prevalence of Angiostrongylus vasorum, Aelurostrongylus abstrusus and Crenosoma vulpis in Native Terrestrial Slug Populations
In each Federal State, two main collection sites were sampled and in each sampling area, four different sites were probed in summer and autumn. Three of four investigated counties (i.e., Obrigheim, Walldürn and Bad Brückenau) were identified as positive regions for lungworm larvae-infected slugs. In Lohr am Rhein (Bavaria), positive gastropods were not detected at any time point.
Of 517 collected slugs, microscopic analysis revealed 81 lungworm larvae-positive samples. Those were analyzed for mono-and co-infections with the different lungworm species via microscopic and molecular analyses. Not shown in this figure are the slugs with non-identifiable metastrongyloid infections (15/81).
Interestingly, seasonal differences in gastropod lungworm prevalence were observed which seemed sampling site-dependent. Hence, an outstanding rise in A. vasorum prevalence from summer to autumn was detected in the county of Obrigheim in Baden- The overall most frequent parasite species found was A. vasorum, with a total prevalence of 11.61% (60/517) (Table 1). Conversely, a lower prevalence was detected for A. abstrusus with 5.65% (9/517), for T. brevior with 0.97% (5/517) and for C. vulpis with 0.77% (4/517). Furthermore, 2.90% (15/517) of the investigated slugs harbored metastrongyloid larvae which could not be identified microscopically to species level due to destroyed morphological relevant structures, i.e., damage at the posterior and/or anterior extremity or cuticle.
Of 517 collected slugs, microscopic analysis revealed 81 lungworm larvae-positive samples. Those were analyzed for mono-and co-infections with the different lungworm species via microscopic and molecular analyses. Not shown in this figure are the slugs with non-identifiable metastrongyloid infections (15/81).
Interestingly, seasonal differences in gastropod lungworm prevalence were observed which seemed sampling site-dependent. Hence, an outstanding rise in A. vasorum prevalence from summer to autumn was detected in the county of Obrigheim in Baden-Wuerttemberg. Thus, within a few months, A. vasorum prevalence increased by more than 4.5-fold from 13.60% in summer to 62.96% in autumn (Table 1). At this sampling site, the prevalence of A. abstrusus also increased within these two seasons by more than 10-fold from 0.80 to 11.11% (Table 1). At the other sampling sites, no consistency in seasonal changes was observed.
Overall, the mean slug weight was 3.00 g and the mean larval burden was 25.48 larvae/slug and 10.12 larvae/g slug tissue, respectively.
Lungworm larvae-positive gastropods all were Arion spp. slugs (92/93), and with one exception, the only collected L. maximus was also harbouring A. vasorum-L3. In total, 67.90% (55/81) of metastrongyloid larvae-positive gastropods hosted ≤ 10 larvae, 27.20% (22/81) were infected with 11-100 larvae, and four (4.90%) showed a high larval burden of > 100, but less than 1000 larvae ( Figure 2). The overall highest larval burden was observed in an Arion spp. slug carrying 802 larval stages, collected in Obrigheim in autumn. We furthermore observed seasonal differences in larval burden and slug weight. Accordingly, during summer, the slugs were infected with a lower larval burden than in autumn, where more gastropods were carrying more than 10 larvae/slug ( Figure 2). Likewise, the mean larval burden per g slug tissue increased from 7.98/g in summer to 12.80/g in autumn. Overall, the mean slug weight was 3.00 g and the mean larval burden was 25.48 larvae/slug and 10.12 larvae/g slug tissue, respectively.

Discussion
The current epidemiological study confirms that various common terrestrial slugs, such as A. vulgaris, A. rufus, D. reticulatum and L. maximus indeed act as competent intermediate hosts for metastrongyloid lungworms in southern parts of Germany [34]. Of high interest, in the county Obrigheim (Baden-Wuerttemberg), a hyperendemic focus for A. vasorum was identified with a prevalence of 62.96% in slug populations in autumn. In contrast, at Bavarian sampling sites, gastropod A. vasorum prevalence in general proved low, showing a maximum value of 0.89%, thereby confirming the well-known patchy distribution of A. vasorum infections in high and low endemic geographic regions. In line with this, other epidemiological studies on gastropod intermediate hosts also showed sampling site-dependent variations of lungworm prevalence ranging from 1.6% to 43% in terrestrial slugs, and were also reported on a classical patchy distribution pattern in this host type [36,[40][41][42]. Likewise, A. vasorum prevalence in domestic and wild canid definitive hosts in Germany were also characterized by various hyperendemic foci strongly varying from 8.4% to 27.3% within different Federal States [35,39].
Furthermore, referring to the hyperendemic focus in Obrigheim, the gastropod prevalence was rising remarkably within months with the season, showing a more than fourtimes higher A. vasorum prevalence in slugs in autumn when compared to summer-related findings. Obviously, in these areas dogs are at a very high risk of acquiring angiostrongylosis when engorging these slugs. These findings reinforce the necessity of including gastropod intermediate host populations in the study and discussion on the spread and emergence of canine angiostrongylosis in Europe, which was reported in the last decades [1,5,23,34,43]. Currently, the underlying causes of the spread of canine angiostrongylosis are not fully understood, and multiple factors, which are regionally distinct and highly context-dependent, are considered to play a role [1]. Thus, environmental parameters, such as climatic factors, edaphic composition and biodiversity with area-specific gastropod populations, consisting of a diversity of species, which also vary e.g., in their life cycles, behaviour, innate immune reactions and lungworm susceptibility, are leading to regionally varying prevalences [1,40,[43][44][45][46][47][48].
In the current study, the invasive Spanish slug (A. vulgaris) and the large red slug (A. rufus) were the most frequently collected gastropod species. These two species have a similar phenotype and can only be differentiated morphologically as juveniles or by dissection of the reproductive tract [49,50]. Arion vulgaris is currently the predominant slug in Germany and is considered as a pest species [51]. Nonetheless, a recent study indicated that this species is indeed native to Western Germany and by mistake was considered to be a neozoan species [51,52]. Due to high slug reproductive rates and beneficial climatic and environmental conditions, this species experienced a massive increase in population and therefore led to the impression of it being an invader rather than a native slug [51]. Members of the family Arionidae are considered as facultative carnivores, showing coprophagic behaviour and are carrion-feeding slugs [53,54]. All slug species investigated in the current study (Arion spp., L. maximus and D. reticulatum) are versatile opportunist feeders. Thus, agonistic behaviour towards conspecifics as well as other species, including cannibalism, has been reported [55][56][57]. Therefore, slug lungworm infections can occur via consumption of larvae present in both the faeces of definitive hosts and the tissue of dead slugs (intermediesis) [58,59]. Moreover, all analyzed slug species are known to be susceptible to metastrongyloid lungworm infections, as previously demonstrated [23,34,46,60]. In the only other epidemiological study in German geographic areas on lungworm infections in gastropods, A. vulgaris also represented the most collected species and the one with the highest lungworm infection rates [36].
Arion vulgaris, A. rufus and D. reticulatum in general have an annual life cycle [61], but semivoltine life cycles can occur as well [46,60]. In contrast, the multivoltine and iteroparous species L. maximus has a longer lifespan of approximately three years. Given that Arionidae grow constantly and lifelong, the collection of adult slugs weighing more than 6 g in summer might indicate that some slugs survived during winter by hibernation or that they were the first ones hatching in autumn of the previous year [47,55,62]. In some studies, gastropod weight and larval burden showed positive correlation [42], while in others, as well as in the current study, there was no statistically significant correlation [40]. Furthermore, the slugs, which weighed more than 6 g, showed rather low larval burdens (Figure 1).
Considering seasonal influences, we documented that the larval burden/slug was higher in autumn than in summer, thereby increasing the risk of definitive host infection in a season-dependent manner. Reinfections of slugs over time and a higher intake capacity of older slug on faeces might represent influencing factors. Of note, the A. vasorum-related prevalence peak in dogs refers to the winter months [35,63]. Considering the prepatent period of four to eight weeks of this parasite, the high prevalence in gastropods in autumn may directly be linked to high prevalence in dogs in winter. Conversely, Lange et al. [36] detected the highest A. vasorum prevalence in German slugs in the summer season. Similar to other European epidemiological studies, Arion spp. slugs were found in greater numbers on collection sites [23,34,40,42,64]. Other slug species like L. maximus and D. reticulatum were also collected, but found in much lower abundance in current sampling areas. Plausible reasons could be their smaller size, less eye-catching colour or general lower occurrence in these geographic locations. Based on the current small sample size of slug species other than Arion spp., it remains speculative whether these species are less infected with lungworms or not. Of note, some studies suggest that Arion spp. acts as a preferred intermediate host [36,42].
It is worthwhile to mention that the climate conditions for the year 2018 of gastropod collection were much warmer and drier than previous year meteorological records in these regions [62]. The influence of climatic conditions, such as extreme drought, on slug populations and metastrongyloid lungworm infections has been controversially discussed [1,32,34,45,49,65]. As such, temperature-driven influences on gastropod populations could be the following. In general, terrestrial slugs respond to changing temperatures by an increase in their daily activities [66]. This phenomenon can often be observed after rainfall at daytime, when temperatures are falling quickly. However, temperature rises above 21 • C stimulate slug activity as well [66]. Obviously, the likelihood of lungworm transmission from the intermediate host to the definitive host will rise when slugs become more active and are thereby exposed to dogs or other hosts instead of staying in their natural hideouts. Thus, changes in temperature might cause an increase of slug activity during the dawn hours and eventually also lead to an enhancement in coprophagic/carrion-feeding behaviours. Moreover, higher environmental temperatures were also documented to positively influence the development of A. vasorum and A. abstrusus larvae within infected gastropods [45,48].
The current study showed that the dynamics of A. vasorum prevalence is fast-changing and highly fluctuating. Hence, data on lungworm prevalence have to be interpreted with care since rapid changes might be seen. Assuming that the spread of A. vasorum can be triggered by different factors, such as intermediate host population dynamics and optimal environmental conditions, any means of early intervention to interrupt the parasite's life cycle in regions where it has not yet disseminated into other intermediate host populations seems beneficial to combat further spreading. However, it can be challenging to identify early lungworm infections in dogs and cats, since clinical signs vary strongly and subclinical cases are common [3,11,64,65,67]. Hence, lungworm infections are easily overseen by both owners and veterinarians. Consequently, veterinary health staff need to be aware of the importance of prophylaxis, preventive treatments and routinely performed screening tests [1,68]. Moreover, pet owners can play an important role in reducing the spread of lungworm infections by their hygiene practices in collecting dog and cat faeces, whenever possible. The fact that pet owners routinely travel with their companion animals to nonendemic areas clearly rises the risk of geographical parasite transmission. Likewise, the closely related feline neuro-angiotropic metastrongyloid parasite Gurltia paralysans, which was originally exclusively reported as endemic in South America [69,70], has recently been recorded in a cat in Spain, presumably due to the import of undiagnosed G. paralysansinfected cats to Europe [71].
As a take-home message, future lungworm-related studies should comprise longterm investigations and include both gastropod population-based estimations and faecal analyses on wild definitive hosts (e.g., foxes, wolves, wild cats or lynxes) in the same regions [4,13,39,72,73]. Moreover, interdisciplinary approaches are required to resolve the complex relationships between the gastropod intermediate host, intermediate host population dynamics, intermediate host species spectrum, paratenic host, climate, environment and metastrongyloid lungworm infections in domestic and wild definitive hosts.

Gastropod Sampling
Overall, 517 slugs were analyzed, with 393 specimens being collected in the summer season and 124 in autumn of 2018. In total, 328 slugs originated from Baden-Wuerttemberg and 189 slugs originated from Bavaria. Sampling areas with reported prevalence for A. vasorum were chosen based on previously published data [35]. Other criteria for the selection of sampling sites included forested landscape and areas with opened grassland, the presence of water bodies such as streams, and proximity to sub-urban human settlements (potential utilization for dog walking). Within these selected locations, we assumed a high potential/probability for metastrongyloid lungworm infections, since foxes, gastropods and companion animals would share the same environment. Thus, four different main areas/cities (Figures 3 and 4), two in Bavaria (Bad Brückenau: 50 •  G. paralysans-infected cats to Europe [71]. As a take-home message, future lungworm-related studies should comprise longterm investigations and include both gastropod population-based estimations and faecal analyses on wild definitive hosts (e.g., foxes, wolves, wild cats or lynxes) in the same regions [4,13,39,72,73]. Moreover, interdisciplinary approaches are required to resolve the complex relationships between the gastropod intermediate host, intermediate host population dynamics, intermediate host species spectrum, paratenic host, climate, environment and metastrongyloid lungworm infections in domestic and wild definitive hosts.

Gastropod Sampling
Overall, 517 slugs were analyzed, with 393 specimens being collected in the summer season and 124 in autumn of 2018. In total, 328 slugs originated from Baden-Wuerttemberg and 189 slugs originated from Bavaria. Sampling areas with reported prevalence for A. vasorum were chosen based on previously published data [35]. Other criteria for the selection of sampling sites included forested landscape and areas with opened grassland, the presence of water bodies such as streams, and proximity to sub-urban human settlements (potential utilization for dog walking). Within these selected locations, we assumed a high potential/probability for metastrongyloid lungworm infections, since foxes, gastropods and companion animals would share the same environment. Thus, four different main areas/cities (Figures 3 and 4  Since slugs are more active at dawn, early in the morning and during temperature changes, sampling was adapted to these conditions and conducted on days of forecasted Since slugs are more active at dawn, early in the morning and during temperature changes, sampling was adapted to these conditions and conducted on days of forecasted rainfall, starting at 6:00 a.m. for approximately 3 h. In total, 517 slugs (393 in summer and 124 in autumn) of four different species (A. rufus, A. vulgaris, D. reticulatum, L. maximus) were collected by hand (Figure 4).
According to current national animal protection laws of Germany, permission for gastropod collection or their use for basic research purposes is not required.

Processing of Gastropod Samples
Each slug was morphologically identified based on characteristics according to [49]. Slugs were cryo-euthanized and stored at −20 • C until further investigation [41]. Gastropod samples were processed as described before [36,41] via artificial HCl/pepsin digestion. Thereafter, in order to remove any undigested material, the samples were sieved twice through a 300 µm and a 25 µm-metal sieve (Retsch, Haan, Germany) according to Segeritz et al. [25]. Remnants of the last sieving process were examined via an optical microscope (Olympus BH-2 ®) , equipped with a digital camera (SC30 ® , Olympus, Tokyo, Japan) at 40×, 200× and 400× magnification. The morphological characteristics of larvae were documented by digital photography, and larvae were counted and carefully collected by pipetting (Pasteur pipette, Hirschmann GmbH & Co. KG, Heilsbronn, Germany). The larvae were stored at 4 • C for further examination.  (Figure 4). According to current national animal protection laws of Germany, permission for gastropod collection or their use for basic research purposes is not required.

Processing of Gastropod Samples
Each slug was morphologically identified based on characteristics according to [49]. Slugs were cryo-euthanized and stored at −20 °C until further investigation [41]. Gastropod samples were processed as described before [36,41] via artificial HCl/pepsin digestion. Thereafter, in order to remove any undigested material, the samples were sieved twice through a 300 µm and a 25 µm-metal sieve (Retsch, Haan, Germany) according to Segeritz et al. [25]. Remnants of the last sieving process were examined via an optical microscope (Olympus BH-2 ®) , equipped with a digital camera (SC30 ® , Olympus, Tokyo, Japan) at 40×, 200× and 400× magnification. The morphological characteristics of larvae were documented by digital photography, and larvae were counted and carefully collected by pipetting (Pasteur pipette, Hirschmann GmbH & Co. KG, Heilsbronn, Germany)., The larvae were stored at 4 °C for further examination.

DNA-Based Confirmation of Lungworm Species
DNA from pooled metastrongyloid larvae from one slug was isolated by using a commercial kit (Quiagen DNeasy Blood and Tissue Kit ® ) and analyzed as reported previously [26,36]. Molecular analyses were performed by conventional PCRs using the nematode forward primer NC1 5 ACGTCTGGTTCAGGGTTGTT-3 and the reverse primers NC2 5 -TTAGTTTCTTTTCCTCCGCT-3 and MetR 5 -CCGCTAAATGATATGCTTA-3 [73,78]. Thereafter, direct sequencing was performed by sending DNA amplicons of samples (n = 12) to a commercial service (LGC Genomics, Berlin, Germany). Resulting sequences were processed by the software Chromas (version 2.6.6) and consensus sequences were compared with sequences deposited in GenBank via the BLAST algorithm (http://www. ncbi.nlm.nih.gov/BLAST, accessed on 15 April 2022).

Conclusions
To the best of our knowledge, this is the first study on the prevalence of metastrongyloids in obligate intermediate hosts in southern parts of Germany, i.e., in the Federal States of Bavaria and Baden-Wuerttemberg. Thereby, the remarkable rise in the prevalence in slugs of A. vasorum within a period of a few months at one location demonstrated the complexity of A. vasorum epidemiology and confirmed the presence of hyperendemic foci in Germany. Moreover, metastrongyloid prevalences in the genus Arion confirmed its role as a pivotal intermediate host of canine angiostrongylosis/crenosomosis as well as feline aelurostrongylosis/troglostrongylosis in Germany. Overall, an interdisciplinary approach in future research projects is required in order to evaluate the complex relationship between gastropod populations, paratenic hosts, climate, environment and lungworm infections. Moreover, veterinary health staff and pet owners should be aware of preventive means and pursue the early treatment of lungworm infections in definitive hosts. Funding: This research was partially financially supported by Bayer Animal Health GmbH, Leverkusen, Germany and was also funded by the Institute of Parasitology, Justus Liebig University Giessen, Germany.

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.