The rat lungworm Angiostrongylus cantonensis is a zoonotic metastrongyloid nematode, currently considered an emerging pathogen approaching Europe. In tropics and subtropics, it is an important food-borne neurotropic parasite of medical and veterinary importance. Sources of infection for mammals and birds include gastropod intermediate hosts and poikilothermic vertebrates (paratenic hosts). To evaluate the relevance of reptiles in the rat lungworm circulation, we performed an experimental series focused on long-term survival of third stage larvae (L3) of A. cantonensis in reptiles and potential of saurians to serve as a source of infection for further hosts. Twenty leopard geckos (Eublepharis macularius) were infected with varying doses of L3 (100, 1000, 10 000 larvae per animal). Live L3 were collected from all infected geckos (mostly in musculature and liver) euthanized 1-6 months after the infection and were proven to be infective for Wistar rats (definitive hosts). Three sacrificed geckos were subsequently fed to three corn snakes (Pantheropis guttatus) to test hypothesis of L3 infectivity for predators positioned higher in the food chain. Snakes were euthanized 1 month post-infection and live L3 were detected predominantly in the intestinal wall. The animals remained clinically healthy throughout the study. No reptiles showed significant changes in haematological and biochemical blood parameters, though elevated CK and GLDH were observed in most geckos in the group receiving higher infectious dose. This study highlights the significant potential of reptiles to play a crucial role in the circulation of metastrongyloid nematodes in food web and in their transmission to humans.

• Klíčová slova
• Angiostrongylus cantonensis, intermediesis, paratenic host, rat lungworm, reptiles,
• MeSH
• Angiostrongylus cantonensis * fyziologie   MeSH
• hadi parazitologie   MeSH
• infekce hlísticemi řádu Strongylida * parazitologie   přenos   veterinární   MeSH
• ještěři * parazitologie   MeSH
• krysa rodu Rattus MeSH
• larva fyziologie   MeSH
• potkani Wistar MeSH
• potravní řetězec * MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

The rat lungworm Angiostrongylus cantonensis is a zoonotic metastrongyloid nematode currently considered an emerging pathogen. Originating in Southeast Asia, this nematode has spread to tropical and subtropical parts of the world via its invasive rodent and gastropod hosts.On the island of Tenerife in the Canary archipelago, the A. cantonensis invasion was recognized more than a decade ago. The endemic lizard Gallotia galloti has been identified as a paratenic host of this nematode in the Canary Island ecosystem. Because this lizard species is the most abundant reptile in Tenerife, we tested its suitability as a possible sentinel for A. cantonensis presence. Lizards were captured alive in nine localities, spanning an environmental gradient across the island. Tail muscle tissue was obtained by provoked caudal autotomy and tested for the nematode infection by a species-specific qPCR. Infection intensities were assessed by detecting A. cantonensis DNA quantities based on a calibrated standard curve. Of the 129 samples tested, 31 were positive. The prevalence varied among localities, with the highest (63.6%) recorded in a humid laurel forest. Even though the prevalence in Valle San Lorenzo was the lowest, this is the first record of A. cantonensis from the arid south of Tenerife. Variation in prevalence at different localities was significantly and positively correlated with increasing vegetation cover and negatively correlated with seasonal variability of precipitation, as determined by Spearman correlation coefficients. Fisher's exact test was used to determine the variation in the prevalence of A. cantonensis among adult males, females, and juveniles and showed no significant difference. Also, there was no significant difference in infection intensity between males and females (as determined by GEE-g). We demonstrated that provoking caudal autotomy can be an effective non-lethal method of A. cantonensis mapping in island ecosystems with abundant lizard species, particularly those with a sharp climatic and vegetation gradient, from xeric to humid conditions.

• Klíčová slova
• Angiostrongylus cantonensis, caudal autotomy, lizards, rat lungworm, sentinels,
• MeSH
• Angiostrongylus cantonensis * izolace a purifikace   MeSH
• infekce hlísticemi řádu Strongylida * epidemiologie   veterinární   parazitologie   MeSH
• ještěři * parazitologie   MeSH
• prevalence MeSH
• sentinelové organismy parazitologie   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Španělsko epidemiologie   MeSH

The metastrongyloid nematode Angiostrongylus cantonensis causes eosinophilic meningitis in a variety of homeothermic hosts including humans. Third-stage infectious larvae develop in gastropods as intermediate hosts. Humans are usually infected by intentional or incidental ingestion of an infected mollusk or paratenic host (poikilothermic vertebrates and invertebrates). The infection may also hypothetically occur through ingestion of food or water contaminated by third-stage larvae spontaneously released from gastropods. Larvae are thought to be released in greater numbers from the intermediate host exposed to stress. This study aimed to compare larval release from stressed with unstressed gastropods. Experimentally infected Limax maximus and Lissachatina fulica were exposed to a stress stimulus (shaking on an orbital shaker). The mucus was collected before and after the stress and examined microscopically and by qPCR for the presence of A. cantonensis larvae and their DNA. In the case of L. maximus, no larvae were detected microscopically in the mucus, but qPCR analysis confirmed the presence of A. cantonensis DNA in all experimental replicates, without clear differences between stressed and non-stressed individuals. In contrast, individual larvae of A. cantonensis were found in mucus from Li. fulica after stress exposure, which also reflects an increased number of DNA-positive mucus samples after stress. Stress stimuli of intensity similar to the transport or handling of mollusks can stimulate the release of larvae from highly infected intermediate hosts. However, these larvae are released in small numbers. The exact number of larvae required to trigger neuroangiostrongyliasis is unknown. Therefore, caution is essential when interacting with potential intermediate hosts in regions where A. cantonensis is endemic.

• Klíčová slova
• Angiostrongylus cantonensis, Gastropods, Larvae release, Stress stimuli,
• MeSH
• Angiostrongylus cantonensis * fyziologie   MeSH
• fyziologický stres * MeSH
• hlen MeSH
• infekce hlísticemi řádu Strongylida parazitologie   MeSH
• kvantitativní polymerázová řetězová reakce MeSH
• larva * fyziologie   MeSH
• plži parazitologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

The rat lungworm Angiostrongylus cantonensis is a metastrongyloid nematode that causes neurological disorders in its accidental hosts, including humans. This invasive pathogen is native to Southeast Asia and adjacent regions and is gradually expanding its distribution to tropical and subtropical areas with new foci discovered near temperate regions. The parasite has a complex life cycle with a range of gastropods serving as intermediate hosts. A broad spectrum of poikilotherm vertebrates and invertebrates can serve as paratenic hosts. Since it has already been demonstrated that other, non-zoonotic metastrongyloids can survive in their intermediate hosts during the winter, the aim of our study was to evaluate the survival of A. cantonensis third-stage larvae in experimentally infected slugs (Limax maximus) kept at 4.5–7°C for 60 days. Third-stage larvae of A. cantonensis survived the period of low temperature and remained capable of infecting definitive hosts (laboratory rats) afterwards, even though their numbers dropped significantly. These results suggest that further spread to higher latitudes or altitudes is possible in areas with sufficient abundance of definitive hosts, since low winter temperatures are not necessarily an obstacle to the spread of the parasite.

• Klíčová slova
• Angiostrongylus cantonensis, Limax maximus, invasive nematode, overwintering,
• MeSH
• Angiostrongylus cantonensis * MeSH
• Angiostrongylus * MeSH
• hlemýždi parazitologie   MeSH
• infekce hlísticemi řádu Strongylida * veterinární   parazitologie   MeSH
• krysa rodu Rattus MeSH
• larva MeSH
• lidé MeSH
• roční období MeSH
• stadia vývoje MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND: Angiostrongylus cantonensis (rat lungworm) is recognised as the leading cause of human eosinophilic meningitis, a serious condition observed when nematode larvae migrate through the CNS. Canine Neural Angiostrongyliasis (CNA) is the analogous disease in dogs. Both humans and dogs are accidental hosts, and a rapid diagnosis is warranted. A highly sensitive PCR based assay is available but often not readily accessible in many jurisdictions. An alternative DNA amplification assay that would further improve accessibility is needed. This study aimed to assess the diagnostic utility of a newly designed LAMP assay to detect DNA of globally distributed and invasive A. cantonensis and Angiostrongylus mackerrasae, the other neurotropic Angiostrongylus species, which is native to Australia. METHODOLOGY/PRINCIPAL FINDINGS: Cerebrospinal fluid (CSF) from dogs with a presumptive diagnosis of A. cantonensis infection (2020-2022) were received for confirmatory laboratory testing and processed for DNA isolation and ultrasensitive Angiostrongylus qPCR targeting AcanR3390. A newly designed LAMP assay targeting the same gene target was directly compared to the reference ultrasensitive qPCR in a diagnostic laboratory setting to determine the presence of A. cantonensis DNA to diagnose CNA. The LAMP assay (Angie-LAMP) allowed the sensitive detection of A. cantonensis DNA from archived DNA specimens (Kappa = 0.81, 95%CI 0.69-0.92; n = 93) and rapid single-step lysis of archived CSF samples (Kappa = 0.77, 95%CI 0.59-0.94; n = 52). Only A. cantonensis DNA was detected in canine CSF samples, and co-infection with A. mackerrasae using amplicon deep sequencing (ITS-2 rDNA) was not demonstrated. Both SYD.1 and AC13 haplotypes were detected using sequencing of partial cox1. CONCLUSIONS/SIGNIFICANCE: The Angie-LAMP assay is a useful molecular tool for detecting Angiostrongylus DNA in canine CSF and performs comparably to a laboratory Angiostrongylus qPCR. Adaptation of single-step sample lysis improved potential applicability for diagnosis of angiostrongyliasis in a clinical setting for dogs and by extension, to humans.

• MeSH
• Angiostrongylus cantonensis * genetika   MeSH
• Angiostrongylus * genetika   MeSH
• diagnostické techniky molekulární MeSH
• hlemýždi genetika   MeSH
• infekce hlísticemi řádu Strongylida * diagnóza   veterinární   MeSH
• krysa rodu Rattus MeSH
• lidé MeSH
• meningitida * diagnóza   veterinární   MeSH
• psi MeSH
• ribozomální DNA MeSH
• techniky amplifikace nukleových kyselin MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• psi MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• ribozomální DNA MeSH

BACKGROUND: Angiostrongylus cantonensis (Ac), or the rat lungworm, is a major cause of eosinophilic meningitis. Humans are infected by ingesting the 3rd stage larvae from primary hosts, snails, and slugs, or paratenic hosts. The currently used molecular test is a qPCR assay targeting the ITS1 rDNA region (ITS1) of Ac. METHODS: In silico design of a more sensitive qPCR assay was performed based on tandem repeats predicted to be the most abundant by the RepeatExplorer algorithm. Genomic DNA (gDNA) of Ac were used to determine the analytical sensitivity and specificity of the best primer/probe combination. This assay was then applied to clinical and environmental samples. RESULTS: The limit of detection of the best performing assay, AcanR3990, was 1 fg (the DNA equivalent of 1/100 000 dilution of a single 3rd stage larvae). Out of 127 CDC archived CSF samples from varied geographic locations, the AcanR3990 qPCR detected the presence of Ac in 49/49 ITS1 confirmed angiostrongyliasis patients, along with 15/73 samples previously negative by ITS1 qPCR despite strong clinical suspicion for angiostrongyliasis. Intermediate hosts (gastropods) and an accidental host, a symptomatic horse, were also tested with similar improvement in detection observed. AcanR3990 qPCR did not cross-react in 5 CSF from patients with proven neurocysticercosis, toxocariasis, gnathostomiasis, and baylisascariasis. AcanR3990 qPCR failed to amplify genomic DNA from the other related Angiostrongylus species tested except for Angiostrongylus mackerrasae (Am), a neurotropic species limited to Australia that would be expected to present with a clinical syndrome indistinguishable from Ac. CONCLUSION: These results suggest AcanR3990 qPCR assay is highly sensitive and specific with potential wide applicability as a One Health detection method for Ac and Am.

• Klíčová slova
• Angiostrongylus, PCR, eosinophilia, meningitis,
• MeSH
• Angiostrongylus cantonensis * genetika   MeSH
• Angiostrongylus * MeSH
• infekce hlísticemi řádu Strongylida * diagnóza   MeSH
• koně MeSH
• krysa rodu Rattus MeSH
• lidé MeSH
• meningitida * MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

In order to elucidate the infection pathways of third stage larvae (L3) of Angiostrongylus cantonensis, we performed experiments to assess: (i) the shedding of L3 from two species of experimental veronicellid slugs drowned in water and the ratio of emerged larvae, (ii) the transmission of viable L3 from drowned terrestrial gastropods to aquatic snails, and (iii) the transmission of viable L3 between terrestrial snails. Molluscs were experimentally infected by first stage larvae (L1) of A. cantonensis. Significantly more L3 larvae were released from Veronicella cubensis than from Veronicella sloanei. Numerous L3 were observed in the muscular foot, and also in the connective tissue between internal organs. Experimental exposure of P. maculata to L3 of A. cantonensis liberated from other gastropod species led to their infection and the infectivity of larvae after intermediesis was demonstrated by infection of laboratory rats (Rattus norvegicus). The transmission of L3 was observed in three out of four experiment replications and L3 were retrieved from 6 out of 24 Subulina octona snails. The infected synanthropic molluscs represent a key component in the epidemiology of human infections by A. cantonensis. Escape of L3 larvae from bodies of dead snails or slugs and their ability to infect further gastropod hosts (intermediesis) represents a public health risk. Thus, control of molluscs living in peri-domestic environment is an essential part of prevention of human infections.

• Klíčová slova
• Angiostrongylus cantonensis, disease, emerging, experimental infection, intermediesis, zoonosis,
• MeSH
• Angiostrongylus cantonensis růst a vývoj   fyziologie   MeSH
• druhová specificita MeSH
• infekce hlísticemi řádu Strongylida parazitologie   přenos   veterinární   MeSH
• krysa rodu Rattus MeSH
• larva růst a vývoj   fyziologie   MeSH
• plži parazitologie   MeSH
• potkani Wistar MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Angiostrongylus cantonensis causes severe neurological disorders in a wide range of warm-blooded animals, including several avian species. A laboratory isolate of A. cantonensis originating from French Polynesia, genotyped as clade 2, was used to assess the effect of experimental infection in chicken and Japanese quail. Low dose groups of birds were infected orally by 100 L3 larvae, high dose groups by 1500 L3 larvae and the birds in the third group were fed three infected snails, mimicking a natural infection. Clinical signs during the first week after infection, haematology, biochemistry, gross lesions and histology findings were used to assess the pathology of the infection. Some of the infected birds showed peripheral eosinophilia, while mild neurological signs were seen in others. No larvae were observed in serial sections of the central nervous system of infected birds 1 week after infection and no major gross lesions were observed during necropsy; histopathology did not reveal lesions directly attributable to A. cantonensis infection. Our results suggest that galliform birds are not highly susceptible to A. cantonensis infection and open a question of the importance of Galliformes in endemic areas as natural pest control, lowering the number of hosts carrying the infective larvae.

• Klíčová slova
• Angiostrongylus, bird, cantonensis, chicken, experimental, quail,
• MeSH
• Angiostrongylus cantonensis růst a vývoj   fyziologie   MeSH
• Coturnix * MeSH
• infekce hlísticemi řádu Strongylida parazitologie   patologie   veterinární   MeSH
• kur domácí * MeSH
• larva růst a vývoj   fyziologie   MeSH
• nemoci drůbeže parazitologie   patologie   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND: Rats (Rattus spp.) invaded most of the world as stowaways including some that carried the rat lungworm, Angiostrongylus cantonensis, the cause of eosinophilic meningoencephalitis in humans and other warm-blooded animals. A high genetic diversity of A. cantonensis based on short mitochondrial DNA regions is reported from Southeast Asia. However, the identity of invasive A. cantonensis is known for only a minority of countries. The affordability of next-generation sequencing for characterisation of A. cantonensis genomes should enable new insights into rat lung worm invasion and parasite identification in experimental studies. METHODS: Genomic DNA from morphologically verified A. cantonensis (two laboratory-maintained strains and two field isolates) was sequenced using low coverage whole genome sequencing. The complete mitochondrial genome was assembled and compared to published A. cantonensis and Angiostrongylus malaysiensis sequences. To determine if the commonly sequenced partial cox1 can unequivocally identify A. cantonensis genetic lineages, the diversity of cox1 was re-evaluated in the context of the publicly available cox1 sequences and the entire mitochondrial genomes. Published experimental studies available in Web of Science were systematically reviewed to reveal published identities of A. cantonensis used in experimental studies. RESULTS: New A. cantonensis mitochondrial genomes from Sydney (Australia), Hawaii (USA), Canary Islands (Spain) and Fatu Hiva (French Polynesia), were assembled from next-generation sequencing data. Comparison of A. cantonensis mitochondrial genomes from outside of Southeast Asia showed low genetic diversity (0.02-1.03%) within a single lineage of A. cantonensis. Both cox1 and cox2 were considered the preferred markers for A. cantonensis haplotype identification. Systematic review revealed that unequivocal A. cantonensis identification of strains used in experimental studies is hindered by absence of their genetic and geographical identity. CONCLUSIONS: Low coverage whole genome sequencing provides data enabling standardised identification of A. cantonensis laboratory strains and field isolates. The phenotype of invasive A. cantonensis, such as the capacity to establish in new territories, has a strong genetic component, as the A. cantonensis found outside of the original endemic area are genetically uniform. It is imperative that the genotype of A. cantonensis strains maintained in laboratories and used in experimental studies is unequivocally characterised.

• Klíčová slova
• Genetic diversity, Invasive species, Mitochondrial genome, Next-generation sequencing, Rat lungworm, Rattus, cox1,
• MeSH
• Angiostrongylus cantonensis genetika   MeSH
• cyklooxygenasa 1 genetika   MeSH
• fylogeneze MeSH
• genetická variace * MeSH
• genom mitochondriální * MeSH
• genom u helmintů MeSH
• infekce hlísticemi řádu Strongylida parazitologie   MeSH
• krysa rodu Rattus MeSH
• mitochondriální DNA * MeSH
• sekvenční analýza DNA MeSH
• sekvenování celého genomu MeSH
• vysoce účinné nukleotidové sekvenování MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Austrálie MeSH
• Havajské ostrovy MeSH
• Polynésie MeSH
• Španělsko MeSH
• Názvy látek
• cyklooxygenasa 1 MeSH
• mitochondriální DNA * MeSH

The efficacy of the drugs flubendazole, levamisole, and their combination was studied against Angiostrongylus cantonensis infection in mice. The drugs were evaluated on day 15 post infection. The efficacy was assessed by using single-dose and 5-dose treatment schedules. Flubendazole was effective at 25 mg/kg, 50 mg/kg, 100 mg/kg, 200 mg/kg when given each day for five days. Levamisole at a dose rate of 200 mg/kg was completely effective in a single treatment. The combined therapy of levamisole (25 mg/kg) and flubendazole (15 mg/kg) produced 100% efficacy. Both drugs given prior to experimental infection showed no prophylactic activity.

• MeSH
• Angiostrongylus MeSH
• kombinovaná farmakoterapie MeSH
• levamisol terapeutické užití   MeSH
• mebendazol analogy a deriváty   terapeutické užití   MeSH
• myši MeSH
• nematodózy farmakoterapie   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• flubendazole MeSH Prohlížeč
• levamisol MeSH
• mebendazol MeSH