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Background Angiostrongyliasis is a zoonotic parasitic disease caused by the rat lungworm Angiostrongylus cantonensis . The intermediate hosts of A. cantonensis are gastropods, and snail species such as Pomacea canaliculata play a key role in the transmission of human angiostrongyliasis. Detecting A. cantonensis infection in snails is an important component of epidemiological surveillance and the control of angiostrongyliasis. Methods In this study, a new method for diagnosing A. cantonensis infection in gastropods was developed by recovering larvae from the buccal cavity of three snail species. The entire buccal cavity of a snail was extracted, and the tissue was pressed between two microscope slides to observe whether A. cantonensis larvae were present. Our new method was compared with traditional pathogenic detection methods of lung microscopy, tissue homogenization, and artificial digestion. We artificially infected 160 P. canaliculata , 160 Cipangopaludina chinensis , and 160 Bellamya aeruginosa snails with A. cantonensis . Then, the four different detection methods were used to diagnose infection in each snail species at 7, 14, 21, and 28 days post exposure. Results We found no significant difference in the percentages of infected P. canaliculata snails using the four methods to detect A. cantonensis larvae. The radula pressing method had a mean detection rate of 80%, while the lung microscopy (81.3%), tissue homogenization (83.8%), and artificial digestion (85%) methods had slightly greater detection rates. Similarly, the percentages of infected C. chinensis snails that were detected using the radula pressing (80%), tissue homogenization (82.1%), and artificial digestion (83.8%) methods were not significantly different. Finally, the percentages of infected B. aeruginosa snails that were detected using the radula pressing (81.3%), tissue homogenization (81.9%), and artificial digestion (81.4%) methods were not significantly different. These results showed that the radula pressing method had a similar detection rate to traditional lung microscopy, tissue homogenization, or artificial digestion methods. Conclusions This study demonstrates a new method for the qualitative screening of gastropods that act as intermediate hosts of A. cantonensis (and other Angiostrongylus species), provides technical support for the control of human angiostrongyliasis, and furthers research on A. cantonensis . Graphical Abstract

Angiostrongylus vasorum, Crenosoma vulpis and Capillaria aerophila are the most common lungworms of domestic and wild canids. We investigated the short- and long-term lungworm prevalence changes in the Swiss fox population with a focus on A. vasorum. Between 2012 and 2017, lungs and hearts of 533 foxes from north-eastern Switzerland were necropsied and blood samples tested for circulating A. vasorum antigen. Angiostrongylus vasorum prevalence increased steadily from 21.5% in 2012 to 81.8% in 2017. In contrast, C. aerophila and C. vulpis prevalences fluctuated between 41.8 and 74.7%, and 3.6 and 14.9%, respectively. Based on 3955 blood samples collected between 1986 and 2017 from three geographic areas and during four time periods, antigen seropositivity increased from 2.4 to 62.0%. In north-eastern Switzerland, seropositivity was initially low (1.9 and 1.7% in the first two time periods) but increased in the following two decades to 22.2 and 62.0%, respectively. Our findings depict the spectacular expansion of A. vasorum in the past three decades. Regionally, the prevalence in foxes increased 4-fold within 6 years in some regions. This underpins the important role of foxes as reservoir hosts, likely explaining the increasing number of cases of canine angiostrongylosis in Switzerland. Our findings are representative of central Europe and may help anticipating future developments in areas where A. vasorum is present but (still) infrequent.

Angiostrongylus costaricensis is a zoonotic parasitic nematode described for the first time in 1971 by Pedro Morera and Rodolfo Céspedes in Costa Rica. This parasite causes an infection known as abdominal angiostrongyliasis, affecting mainly school-aged children and young adults. Infection with A. costaricensis has been associated with a myriad of rodent and mollusk species in the Americas and the Caribbean, as its natural hosts and reservoirs. In this commemorative review, we highlight the extensive research collected through a 50-year journey, which includes ecological, pathological, and molecular studies on A. costaricensis and its implicated disease. We also identify major knowledge gaps in its evolutionary history, the ecological role of imported and invasive mollusk species, and immune response. We propose that the advent of -omics analyses will allow us to gather novel information regarding A. costaricensis biology and infection dynamics, as well as to promote the design of much-needed sensitive and specific diagnostic tools.

Angiostrongylus cantonensis is a metastrongyloid nematode found widely in the Asia-Pacific region, and the aetiological agent of angiostrongyliasis; a disease characterized by eosinophilic meningitis. Rattus rats are definitive hosts of A. cantonensis, while intermediate hosts include terrestrial and aquatic molluscs. Humans are dead-end hosts that usually become infected upon ingestion of infected molluscs. A presumptive diagnosis is often made based on clinical features, a history of mollusc consumption, eosinophilic pleocytosis in cerebral spinal fluid, and advanced imaging such as computed tomography. Serological tests are available for angiostrongyliasis, though many tests are still under development. While there is no treatment consensus, therapy often includes a combination of anthelmintics and corticosteroids. Angiostrongyliasis is relatively rare, but is often associated with morbidity and sometimes mortality. Recent reports suggest the parasites’ range is increasing, leading to fatalities in regions previously considered Angiostrongylus-free, and sometimes, delayed diagnosis in newly invaded regions. Increased awareness of angiostrongyliasis would facilitate rapid diagnosis and improved clinical outcomes. This paper summarizes knowledge on the parasites’ life cycle, clinical aspects and epidemiology. The molecular biology of Angiostrongylus spp. is also discussed. Attention is paid to the significance of angiostrongyliasis in Australia, given the recent severe cases reported from the Sydney region.

Background Gastropod-borne metastrongyloid lungworm infections are poorly understood despite their importance in both veterinary and human medicine. This study intends to assess the use of nuclear imaging with whole-body positron emission tomography and computed tomography to scan radiolabeled Angiostrongylus vasorum - and Crenosoma striatum first-stage larvae (L1) while migrating in vivo in the obligate mollusc intermediate hosts. Here, the giant African snail ( Lissachatina fulica ) was used as a novel animal model for lungworm-associated investigations, as this gastropod species is known to act as a natural obligate intermediate host in the tropics for various metastrongyloid lungworms, including the zoonotic-relevant Angiostrongylus cantonensis . Radiolabeled A. vasorum - and C. striatum L1 migration was visualized through nuclear imaging after L1 oral infection or injection. Methods Live L1 were collected through the standard Baermann funnel technique. After isolation and assessment of larval viability, collected A. vasorum - and C. striatum L1 were incubated with a radiolabeled glucose analogue, 18 F-fluordesoxyglucose. Thereafter, radiolabeled L1 were fed orally or injected into adult L. fulica , and in vivo scans were performed to visualize larval migration routes at different time points post infection through various gastropod organs/tissues. Results The most optimal incubation time of larvae and 18 F-fluordesoxyglucose was 30 min. After washing nonincorporated tracer, metastrongyloid L1 retained on average activity of 0.33 (0.103) KBq per larvae. This was the maximum activity achieved, and even longer incubation times, i.e., 60 and 120 min, did not exceed this value. The in vivo scans showed dispersal from the site of larval injection or feeding. Radiolabeled A. vasorum - or C. striatum L1 moved rapidly from the site of injection or the oral cavity with nonspecific accumulation in one or numerous gastropod organs at 30 min post infection. Conclusions This study concludes that radiolabeling of metastrongyloid L1 with 18 F-fluordesoxyglucose is achievable up to a level that can be detected in a scan of individual snails. Scanning L1 larvae in L. fulica at 60 min still represents larval migration. After 2 h, this study found that the migration is already widespread, and the activity is too low to be narrowed to a specific organ. Detailed in vivo scans of gastropods with not only higher infectious doses but also other radiolabeled tracers and longer observation periods might allow detection of either organ tropism or larval accumulation in certain mollusc tissues/organs for further development into infective stages. Graphical abstract

Blood contains hundreds of proteins, reflecting ongoing cellular processes and immune reactions. Infections with the blood-dwelling cardiopulmonary nematode Angiostrongylus vasorum in dogs manifest with a broad spectrum of clinical signs including respiratory distress, bleeding diathesis and neurological signs, and are associated with a perturbed blood protein profile in dogs. However, current knowledge does not completely explain the observed pathologies induced by A. vasorum infections, including bleeding disorders. Using sera from experimentally infected dogs, dog serum proteome was analysed by quantitative mass spectrometry methods over several time points before and after inoculation. Following computational analysis, we identified 139 up- and downregulated proteins after infection (log2 ratio cut-off ≥ 1.0; q-value ≤ 0.05). Among upregulated proteins were chitinase 3-like 1 and pulmonary surfactant-associated protein B (log2 fold-changes ≥ 5). Pathway enrichment revealed the complement (especially the lectin pathway) and coagulation cascades as significantly affected upon analysis of downregulated proteins. Among them were mannan-binding lectin serine peptidases, ficolin, and coagulation factor XIII-B. These results bring new elements towards understanding the underlying pathomechanisms of bleeding diatheses observed in some A. vasorum -infected dogs.

Background Metastrongyloid parasites Angiostrongylus vasorum and Crenosoma vulpis infect wild and domestic canids and are important pathogens in dogs. Recent studies indicate that gastropod intermediate hosts infected with various metastrongyloids spontaneously shed infective third-stage larvae (L3) into the environment via feces and mucus under laboratory conditions. Shed L3 retain motility up to 120 days, but whether they retain infectivity was unknown. Methods To assess the infectivity of shed L3, the heart/lungs of six red foxes ( Vulpes vulpes ) were obtained from trappers in Newfoundland, Canada. Lungs were examined for first-stage larvae (L1) by the Baermann technique. A high number of viable A. vasorum L1 and a low number of C. vulpis L1 were recovered from one fox; these were used to infect naïve laboratory-raised Limax maximus . L3 recovered from slugs by artificial digestion were fed to two naïve purpose-bred research beagles (100 L3/dog). L1 shed by these two dogs was used to infect 546 L. maximus (2000–10,000 L1/slug). L3 shedding was induced by anesthetizing slugs in soda water and transferring them into warm (45 °C) tap water for at least 8 h. Shed L3 recovered from slugs were aliquoted on romaine lettuce in six-well tissue culture plates (80–500 L3/well) and stored at 16 °C/75% relative humidity. Four naïve research beagles were then exposed to 100 L3/dog from larvae stored for 0, 2, 4, or 8 weeks, respectively, after shedding. Results All four dogs began shedding C. vulpis L1 by 26–36 days post-infection (PI). All four dogs began shedding A. vasorum L1 by 50 days PI. Conclusions L3 infectivity for the definitive host was retained in both metastrongyloids, indicating the potential for natural infection in dogs through exposure from environmental contamination. As an additional exposure route, eating or licking plant or other material(s) contaminated with metastrongyloid L3 could dramatically increase the number of dogs at risk of infection from these parasites. Graphical Abstract

Climate change is implicated in the alteration of the ranges of species worldwide. Such shifts in species distributions may introduce parasites/pathogens, hosts, and vectors associated with disease to new areas. The parasite Angiostrongylus ( = Parastrongylus) cantonensis is an invasive species that causes eosinophilic meningitis in humans and neurological abnormalities in domestic/wild animals. Although native to southeastern Asia, A. cantonensis has now been reported from more than 30 countries worldwide. Given the health risks, it is important to describe areas with potentially favorable climate for the establishment of A. cantonensis, as well as areas where this pathogen might become established in the future. We used the program Maxent to develop an ecological niche model for A. cantonensis based on 86 localities obtained from published literature. We then modeled areas of potential A. cantonensis distribution as well as areas projected to have suitable climatic conditions under four Representative Concentration Pathways (RCP) scenarios by the 2050s and the 2070s. The best model contained three bioclimatic variables: mean diurnal temperature range, minimum temperature of coldest month and precipitation of warmest quarter. Potentially suitable habitat for A. cantonensis was located worldwide in tropical and subtropical regions. Under all climate change RCP scenarios, the center of the projected distribution shifted away from the equator at a rate of 68-152 km per decade. However, the extent of areas with highly suitable habitat (>50%) declined by 10.66-15.66% by the 2050s and 13.11-16.11% by the 2070s. These results conflict with previous studies, which have generally found that the prevalence of tropical pathogens will increase during the 21st century. Moreover, it is likely that A. cantonensis will continue to expand its current range in the near future due to introductions and host expansion, whereas climate change will reduce the total geographic area of most suitable climatic conditions during the coming decades.

Background Angiostrongylus vasorum is a nematode residing in the heart and pulmonary vessels of dogs and wild carnivores. In Europe the red fox is its reservoir, while only three records from wolves have been published. Angiostrongylus vasorum has a worldwide distribution, and many pieces of evidence demonstrate that it is spreading from endemic areas to new ones. In Italy, A. vasorum was reported with increasing frequency in dogs and foxes in the last decades, and now it is considered endemic throughout the country. Angiostrongylus vasorum can be asymptomatic or cause respiratory and circulatory disorders, at times causing severe disseminated infections. Methods Between February 2012 and December 2016, 25 wolves found dead in central Italy were submitted to the Istituto Zooprofilattico del Lazio e della Toscana for post-mortem examination. Samples of lungs, heart, liver, spleen, kidneys, mediastinic lymph nodes and brain were collected from each animal for histological examination. When adult and larval nematodes were microscopically seen in lungs, the other organs were processed, and five histological sections for each organ were examined. To confirm parasite identification, lung samples were submitted to a PCR-sequencing protocol targeting the ITS2 region of A. vasorum. Results Seven wolves (28.0%) harboured nematode larvae in lung sections. In two of the positive wolves, adult nematodes were visible in pulmonary arteries, in four animals larvae were also detected in other organs. DNA sequencing reactions confirmed parasite identification as A. vasorum in all the cases. Conclusions As a result of the high prevalence of A. vasorum reported in wolves in the present study, a focus of high circulation could be hypothesised in central Italy. Nevertheless, the similarly high prevalence in foxes originating from the same areas were reported in previous papers. Histopathological evidence highlights the pathogenic potential of A. vasorum in the wolf, especially in juvenile animals.

Background Phagocyte-derived extracellular traps (ETs) were recently demonstrated mainly in vertebrate hosts as an important effector mechanism against invading parasites. In the present study we aimed to characterize gastropod-derived invertebrate extracellular phagocyte trap (InEPT) formation in response to larval stages of important canine and feline metastrongyloid lungworms. Gastropod haemocytes were isolated from the slug species Arion lusitanicus and Limax maximus , and the snail Achatina fulica , and exposed to larval stages of Angiostrongylus vasorum , Aelurostrongylus abstrusus and Troglostrongylus brevior and investigated for gastropod-derived InEPT formation. Results Phase contrast as well as scanning electron microscopy (SEM) analyses of lungworm larvae-exposed haemocytes revealed ET-like structures to be extruded by haemocytes thereby contacting and ensnaring the parasites. Co-localization studies of haemocyte-derived extracellular DNA with histones and myeloperoxidase in larvae-entrapping structures confirmed classical characteristics of ETs. In vivo exposure of slugs to A. vasorum larvae resulted in InEPTs being extruded from haemocytes in the slug mucous extrapallial space emphasizing the pivotal role of this effector mechanism against invasive larvae. Functional larval entrapment assays demonstrated that almost half of the haemocyte-exposed larvae were contacted or even immobilized by released InEPTs. Overall, as reported for mammalian-derived ETs, different types of InEPTs were here observed, i.e. aggregated, spread and diffused InEPTs. Conclusions To our knowledge, this study represents the first report on metastrongyloid lungworm-triggered ETosis in gastropods thereby providing evidence of early mollusc host innate immune reactions against invading larvae. These findings will contribute to the better understanding on complex parasite-intermediate host interactions since different gastropod species bear different transmitting capacities for metastrongyloid infections.