Release of Angiostrongylus cantonensis larvae from live intermediate hosts under stress.

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.

Detection of rat lungworm (Angiostrongylus cantonensis) infection by real-time PCR from the peripheral blood of animals: a preliminary study.

Rat lungworm disease or neuroangiostrongyliasis is a cerebral parasitic infection that affects humans and animals alike. Its clinical signs and symptoms can range from mild self-resolving to serious life-threatening conditions. Studies suggest therapeutic interventions during the early stages of infection to be more effective than in later stages. However, early diagnosis of infection is usually problematic without the knowledge of exposure and/or detection of the parasite's DNA or antibody against the parasite in the cerebrospinal fluid. This requires a lumbar puncture, which is an invasive procedure that generally requires hospitalization. This study evaluates an affordable and less invasive alternative to detect parasitic DNA by PCR from the peripheral blood of potentially infected animals. Blood samples from 58 animals (55 dogs and 3 cats) with clinical suspicion of infection were submitted to our lab between February 2019 and August 2022 by local, licensed veterinarians. DNA was extracted from whole blood, plasma, serum, and/or packed cells using the Qiagen DNeasy Blood & Tissue Kit as per the manufacturer's protocol. All 58 animals were tested by real-time PCR using the AcanITS1 assay and 32 of these animals (31dogs; 1 cat) were also tested using the AcanR3990 assay. The PCR results for both assays were classified into strongly positive > positive > weakly positive > negative, and equivocal for ambiguous results, based on the strength of the signal. The percent infection detected using the AcanITS1 and AcanR3990 assays was 12.72% (7/55) and 20.68% (6/29), respectively. The overall percent infection detected was 34.37% (11/32), with only two animals testing positive by both assays. The three cats involved in this study tested negative by both assays. These results are promising and warrant further investigations to increase sensitivity including variables that might affect detection in the blood, such as parasite load, and laboratory methodologies.

Semperula wallacei (Mollusca, Veronicellidae) um hospedeiro natural recém-descoberto de Angiostrongylus cantonensis (Nematoda, Angiostrongylidae) na Bacia do Pacífico.

Semperula wallacei (Issel, 1874) is a species of terrestrial slug that occurs in southeast China and the Pacific Basin and is the only species of its genus that occurs beyond the Oriental region and to the east of Wallace's line in the Australian region, where it has probably been introduced. In this study, we report for the first time S. wallacei as an intermediate host for Angiostrongylus cantonensis (Chen, 1935) based on histological and molecular analyses of slugs from Tuamasaga, Samoa, deposited at the Medical Malacological Collection (Fiocruz-CMM). DNA was obtained from the deparafinized tissues scraped from specimen slides. Polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) targeted to the internal transcribed spacer 2 (ITS2) region were carried out using the restriction enzyme Cla I. The RFLP profile observed for our larval specimen of S. wallacei was identical to the profile previously established for A. cantonensis, demonstrating that S. wallacei can be naturally infected with A. cantonensis and is likely to be an intermediate host for this parasitic nematode species in the field. The potential for geographical range expansion of S. wallacei in the Pacific Basin, its small size, and the general role of veronicellids as crop pests and hosts of nematodes, indicate the significance of S. wallacei as an invasive species in the Pacific Basin. Our work also highlights the importance of biological collections for investigating the environmental impact of invasive species on agriculture, public health, and biodiversity conservation.

Angiostrongylus cantonensis Infection in Brown Rats (Rattus norvegicus), Atlanta, Georgia, USA, 2019-2022.

Rat lungworm (Angiostrongylus cantonensis), a zoonotic parasite invasive to the United States, causes eosinophilic meningoencephalitis. A. cantonensis harbors in rat reservoir hosts and is transmitted through gastropods and other paratenic hosts. We discuss the public health relevance of autochthonous A. cantonensis cases in brown rats (Rattus norvegicus) in Atlanta, Georgia, USA.

CEBPα/miR-101b-3p promotes meningoencephalitis in mice infected with Angiostrongylus cantonensis by promoting microglial pyroptosis.

BACKGROUND: Angiostrongylus cantonensis (A. cantonensis) infection can induce acute inflammation, which causes meningoencephalitis and tissue mechanical injury to the brain. Parasite infection-induced microRNAs play important roles in anti-parasite immunity in non-permissive hosts. miR-101b-3p is highly expressed after A. cantonensis infection; however, the role of miR-101b-3p and the transcription regulation of miR-101b-3p in A. cantonensis infection remain poorly characterized. RESULTS: In the present study, we found that miR-101b-3p inhibition alleviated inflammation infiltration and pyroptosis in A. cantonensis infection. In addition, we found that CCAAT/enhancer-binding protein alpha (CEBPα) directly bound to the - 6-k to - 3.5-k region upstream of miR-101b, and CEBPα activated miR-101b-3p expression in microglia. These data suggest the existence of a novel CEBPα/miR-101b-3p/pyroptosis pathway in A. cantonensis infection. Further investigation verified that CEBPα promotes pyroptosis by activating miR-101b-3p expression in microglia, and microglial pyroptosis further promoted inflammation. CONCLUSIONS: Our results suggest that a CEBPα/miR-101b-3p/pyroptosis pathway may contribute to A. cantonensis infection-induced inflammation and highlight the pro-inflammatory effect of miR-101b-3p. Video Abstract.

Rat lungworm survives winter: experimental overwintering of Angiostrongylus cantonensis larvae in European slugs.

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 4.5­7°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.

Rat lungworm (Angiostrongylus cantonensis) active larval emergence from deceased bubble pond snails (Bullastra lessoni) into water.

Angiostrongylus cantonensis (the rat lungworm) is a zoonotic parasite of non-permissive accidental (dogs, humans, horses, marsupials, birds) hosts. The 3rd stage larvae (L3s) in the intermediate host (molluscs) act as the source of infection for accidental hosts through ingestion. Larvae can spontaneously emerge from dead gastropods (slugs and snails) in water, which are experimentally infective to rats. We sought to identify the time when infective A. cantonensis larvae can autonomously leave dead experimentally infected Bullastra lessoni snails. The proportion of A. cantonensis larvae that emerge from crushed and submerged B. lessoni is higher in snails 62 days post-infection (DPI) (30.3%). The total larval burden of snails increases at 91 DPI, indicating that emerged larvae subsequently get recycled by the population. There appears to be a window of opportunity between 1 and 3 months for infective larvae to autonomously escape dead snails. From a human and veterinary medicine viewpoint, the mode of infection needs to be considered; whether that be through ingestion of an infected gastropod, or via drinking water contaminated with escaped larvae.

Identification and genetic characterization of Angiostrongylus cantonensis isolated from the human eye.

Angiostrongylus cantonensis, or the rat lungworm, is the causative agent of human angiostrongyliasis associated with eosinophilic meningitis or meningoencephalitis. Additionally, this nematode can cause ocular angiostrongyliasis, though this is rare. The worm can cause permanent damage to the affected eye and sometimes even blindness. Genetic characterization of the worm from clinical samples is limited. In the present study, we investigated the genetics of A. cantonensis recovered from a patient's eye in Thailand. We sequenced two mitochondrial genes (cytochrome c oxidase subunit I, or COI, and cytochrome b, or cytb) and nuclear gene regions (66-kDa protein and internal transcribed spacer 2, or ITS2) from a fifth-stage larva of Angiostrongylus sample that was surgically removed from the human eye. All sequences of the selected nucleotide regions were highly similar (98-100%) to the sequences of A. cantonensis in the GenBank database. The maximum likelihood and neighbor-joining trees of the COI gene indicated that A. cantonensis was closely related to the AC4 haplotype, whereas the cytb and 66-kDa protein genes were closely clustered with the AC6 and Ac66-1 haplotypes, respectively. In addition, the phylogeny of the concatenated nucleotide datasets of the COI and cytb revealed that the worm was closely related to the Thai strain and strains from other countries. This study confirms the identification and genetic variation of the fifth-stage larvae of A. cantonensis recovered from a patient's eye in Thailand. Our findings are important for future research on the genetic variation of A. cantonensis that causes human angiostrongyliasis.

Parasitism in Rattus rattus and sympatric Achatina fulica by Angiostrongylus cantonensis in an urban park in southeast Brazil.

In this study, rodents (Rattus rattus) and mollusks (Achatina fulica) were captured in a small forest located in a large metropolitan city in Brazil, and they were examined to investigate possible parasitism by Angiostrongylus cantonensis. The parasites were recovered as helminths from the pulmonary arteries of the synanthropic rodents and as third-stage larvae (with Metastrongylidae family characteristics) from the mollusks. To confirm the species, these larvae were used to experimentally infect Rattus norvegicus for the posterior recovery of adult helminths. To identify the adult helminths, morphological, morphometric, molecular, and phylogenetic techniques were employed. Furthermore, we also characterized the histological lesions associated with parasitism in naturally infected definitive hosts. Our results demonstrated the occurrence of a natural life cycle of A. cantonensis (with the presence of adult helminths) in definitive hosts, Rattus rattus, and third-stage larvae in an intermediate host, A. fulica. In free-living rodents, lesions of granulomatous pneumonia in the lungs and meningitis in the brain were also found. These results warn of the risk of accidental transmission of A. cantonensis to human residents around the park because of the extensive interaction among the fauna of the park, domestic animals, and the surrounding human population.

Application of the Midi Parasep® SF technique for the detection of L1 larvae of Angiostrongylus cantonensis in faeces of infected rats.

We investigated parasitic zoonoses caused by protozoans and helminths in urban and peri-urban rat populations (Rattus norvegicus and R. rattus) in Spanish cities. Rats were trapped and then dissected to remove adult helminths, and the contents of the large intestine were retrieved for the study of parasitic forms. The Midi Parasep® solvent free (SF) technique was used to concentrate the parasites in the intestinal contents. Some of the rats studied (n = 8) were infected by the rat lungworm, Angiostongylus cantonensis, whose first stage larvae (L1) are shed in rat faeces. After the concentration technique, L1 larvae were found in the sediment of 6 of the 8 positive rats. The two negative sediment samples were due to the presence of either only adult females or, in addition to males, only young females in the lungs of the rats. In view of our results, Midi Parasep® SF turned out to be a simple, rapid, inexpensive, and sensitive method to detect nematode larvae, such as the L1 larvae of A. cantonensis (or A. costaricensis), in natural and experimentally infected rats.

Clinical Efficacy and Safety of Albendazole and Other Benzimidazole Anthelmintics for Rat Lungworm Disease (Neuroangiostrongyliasis): A Systematic Analysis of Clinical Reports and Animal Studies.

The safety and efficacy of benzimidazole anthelmintics for the treatment of rat lungworm disease (neuroangiostrongyliasis) have been questioned regardless of numerous experimental animal studies and clinical reports. In this review, 40 of these experimental animal studies and 104 clinical reports are compiled with a focus on albendazole. Among the 144 articles involving an estimated 1034 patients and 2561 animals, 4.1% were inconclusive or vague regarding the use of benzimidazoles. Of the remaining 138 articles, 90.5% found benzimidazoles to be safe and effective (885 patients, 2530 animals), 4.3% as safe but ineffective (73 patients, 3 animals), and 5.0% caused adverse reactions (7 patients, 28 animals). Among those clinical reports that described a confirmed diagnosis of neuroangiostrongyliasis in which albendazole monotherapy was used, 100% reported high efficacy (743 patients, 479 animals). In those where albendazole-corticosteroid co-therapy was used, 97.87% reported it to be effective (323 patients, 130 animals).

Angiostrongylus cantonensis Nematode Invasion Pathway, Mallorca, Spain.

Neural angiostrongyliasis is an emerging zoonosis caused by the rat lungworm, Angiostrongylus cantonensis. In humans, infection with this nematode often results in eosinophilic meningitis and other severe disorders of the central nervous system. Europe was deemed a nonendemic region until 2018, when A. cantonensis worms were detected on the Mediterranean island of Mallorca, Spain, a tourism hotspot. Since that time, a sentinel surveillance system and a molecular approach have been used to follow the invasion path of the rat lungworm on the island. A. cantonensis worms have been found in animals from 8 locations on the island over 3 consecutive years. Our preliminary results show a recognizable pattern of clinical signs in infected hedgehogs and a single mitochondrial haplotype circulating in Mallorca. We present strong evidence confirming that the rat lungworm has successfully established and colonized an island in Europe and discuss observations and possible strategies for its early detection across continental Europe.

Autochthonous Angiostrongylus cantonensis Lungworms in Urban Rats, Valencia, Spain, 2021.

To determine the role of rats as potential reservoirs of zoonotic parasites, we examined rats trapped in urban sewers of Valencia, Spain, in 2021. Morphologic and molecular identification and sequencing identified autochthonous Angiostrongylus cantonensis nematodes, the most common cause of human eosinophilic meningitis, in pulmonary arteries of Rattus norvegicus and R. rattus rats.

Angiostrongylus cantonensis in a Red Ruffed Lemur at a Zoo, Louisiana, USA.

A red ruffed lemur (Varecia rubra) from a zoo in Louisiana, USA, was euthanized for worsening paresis. Brain and spinal cord histology identified eosinophilic meningoencephalomyelitis with intralesional adult Angiostrongylus sp. nematodes. PCR and sequencing confirmed A. cantonensis infection, indicating this parasite constitutes an emerging zoonosis in the southeastern United States.

Synaptic loss and progression in mice infected with Angiostrongylus cantonensis in the early stage.

BACKGROUND: Angiostrongylus cantonensis is also known as rat lungworm. Infection with this parasite is a zoonosis that can cause eosinophilic meningitis and/or eosinophilic meningoencephalitis in humans and may lead to fatal outcomes in severe cases. In this study, we explored the mechanisms of the impairments in the cognitive functions of mice infected with A. cantonensis. METHODS: In infected mice with different infective intensities at different timepoint postinfection, loss and recovery of cognitive functions such as learning and memory abilities were determined. Neuronal death and damage to synaptic structures were analyzed by Western blotting and IHC in infected mice with different infection intensities at different timepoint postinfection. RESULTS: The results of behavioral tests, pathological examinations, and Golgi staining showed that nerve damage caused by infection in mice occurred earlier than pathological changes of the brain. BDNF was expressed on 14 day post-infection. Cleaved caspase-3 increased significantly in the late stage of infection. However, IHC on NeuN indicated that no significant changes in the number of neurons were found between the infected and uninfected groups. CONCLUSIONS: The synaptic loss caused by the infection of A. cantonensis provides a possible explanation for the impairment of cognitive functions in mice. The loss of cognitive functions may occur before severe immunological and pathological changes in the infected host.

Host transmission dynamics of first- and third-stage Angiostrongylus cantonensis larvae in Bullastra lessoni.

Given the importance of angiostrongyliasis as an emerging infectious disease of humans, companion animals, and wildlife, the current study focused on the transmission dynamics of first- and third-stage larvae of the parasitic nematode, Angiostrongylus cantonensis. The migration of infective larvae and their subsequent distribution within the Lymnaeidae snail, Bullastra lessoni, were investigated over time using microscopic examination of histological sections and fresh tissue. Snails were divided into four anatomical regions: (i) anterior and (ii) posterior cephalopedal masses, (iii) mantle skirt and (iv) visceral mass. The viability of free-swimming third-stage larvae, after their release from snail tissues, was evaluated in vitro by propidium iodide staining and infectivity by in vivo infection of Wistar rats. Snails were sequentially dissected over time to assess the number and anatomical distribution of larvae within each snail and hence infer their migration pathway. Herein, ongoing larval migratory activity was detected over 28 days post-infection. A comparison of infection rates and the larval distribution within the four designated snail regions demonstrated a significant relationship between anatomical region and density of infective larvae, with larvae mostly distributed in the anterior cephalopedal mass (43.6 ± 10.8%) and the mantle skirt (33.0 ± 8.8%). Propidium iodide staining showed that free-swimming third-stage larvae retained viability for between 4 and 8 weeks when stored under laboratory conditions. In contrast to viability, larval infectivity in rats remained for up to 2 weeks only. Knowledge gained from the current work could provide information on the development of new approaches to controlling the transmission of this parasite.

Evaluation of the mechanism of action of albendazole on adult rat lungworm (Angiostrongyluscantonensis).

Albendazole is considered the anthelmintic of choice for the management of rat lungworm disease (neuroangiostrongyliasis), due to its broad spectrum of nematocidal activity and its ability to cross the blood-brain barrier. Albendazole binds to ß-tubulins, preventing their polymerization into microtubules, thereby corrupting the cascade of cell division at metaphase, which ultimately leads to the death of individual cells and eventually the death of the parasite. Inhibition of microtubule formation will also hinder the axoplasmic transport system, affecting the neuronal activities of the parasite. While this mechanism has been explicated in other parasitic and non-parasitic nematodes, it has never been evaluated in Angiostrongylus cantonensis. This study evaluates the antimitotic effects of albendazole sulphoxide (active metabolite) on the microtubules of adult A. cantonensis using the tubulin polymerization assay and measures its effects on worm viability using the colorimetric MTT assay. Three different concentrations of albendazole (62.5 µM, 250 µΜ, and 1 mM) were evaluated. We saw a statistically significant dose-dependent reduction in the band intensity of polymerized tubulins (or microtubules) (P = 0.019), suggesting that albendazole imparts its antimitotic effect in a dose-dependent manner. Similarly, our MTT assay showed a dose-dependent decrease in formazan intensity (proportional to cell viability), suggesting that the rate of nematocidal activity of albendazole is also proportional to its concentration. In compiling the results from both these experiments, a correlation between the microtubule assembly and worm viability is evident.

Immunoanalysis of different antigenic preparations of Angiostrongylus cantonensis for neuroangiostrongyliasis diagnosis improvement.

BACKGROUND: Angiostrongylus cantonensis is the etiological agent of neuroangiostrongyliasis in humans, which is developed in gastropods and vertebrate species, mainly rodents. Human transmission occurs through consumption of molluscs and paratenic hosts infected with L3, and the migration of larvae to the central nervous system causes eosinophilic meningitis. Laboratory diagnosis is based on molecular and immunological tests, using young or adult females as a source of antigens. However, these tests give positive results only after several weeks of symptoms onset and also cross-reactions with others parasite infections may occur. OBJECTIVES: The purpose of this work was to study different antigenic preparations of distinct evolutionary phases of A. cantonensis, in order to improve serological techniques for disease immunodiagnosis. METHODS: For this purpose, antigenic fractions of different evolutionary forms were evaluated by Dot-enzyme-linked immunosorbent assay (Dot-ELISA) and Western blot using serum samples. FINDINGS: All analysed fractions showed reactivity with serum samples from patients with neuroangiostrongyliasis, especially female membrane alkaline (FAM) and female soluble alkaline (FAS) fractions together with female soluble saline (FSS), improving the technique specificity. MAIN CONCLUSIONS: The results point to the possibility of use of raw female antigens in association with alkaline membrane antigens extracted from adult worms to aid in diagnosis and helps initiate neuroangiostrongyliasis surveillance and control actions.

Eosinophilic meningoencephalitis caused by rat lungworm (Angiostrongylus cantonensis) migration in a white-eared opossum (Didelphis albiventris) with concurrent distemper virus in southern Brazil.

Angiostrongylus cantonensis is a worldwide zoonotic parasite that causes eosinophilic meningoencephalitis in many species of animals including humans. This report describes neuro-angiostrongylosis in a white-eared opossum that showed nervous clinical signs such as circling and depression. At necropsy, no relevant macroscopic lesions were observed. Histologically, eosinophilic meningoencephalitis was associated with multiple sections of nematodes and many intracytoplasmic eosinophilic inclusion bodies within gastric parietal cells. Immunohistochemistry was strongly positive for canine distemper virus in the stomach but there was no immunolabeling in the brain. This study describes a fatal case of eosinophilic meningoencephalitis by A. cantonensis with canine distemper virus concurrent infection in a white-eared opossum in southern Brazil, with histological characterization and molecular confirmation of the parasitism.

The invasive giant African land snail, Achatina fulica (Gastropoda: Pulmonata): global geographical distribution of this species as host of nematodes of medical and veterinary importance.

The giant African land snail, Achatina fulica, is an important invasive species in many countries, where it causes losses in biodiversity and agriculture, as well as impacting the health of both humans and animals, as the intermediate host of medically important nematodes. The present study is based on a comprehensive review of the literature on the nematodes that have been found in association with A. fulica, worldwide. We searched a number of different databases and used the findings to investigate the methods used to extract and identify the nematodes, their larval stages, and environment and collecting procedures of the infected molluscs. Between 1965 and 2021, 11 nematode species were recorded in association with A. fulica in 21 countries. Most of the studies recorded associations between A. fulica and Angiostrongylus cantonensis, which causes cerebral angiostrongyliasis in humans and Aelurostrongylus abstrusus, which provokes pneumonia in felines. The nematodes were extracted primarily by artificial digestion with hydrochloric acid or pepsin, and identified based on their morphology or through experimental infection to obtain the adult. In most cases, the nematodes were at larval stage L3, and the infected A. fulica were collected from anthropogenic environments. The results demonstrate the importance of A. fulica as a host of nematodes of medical and veterinary importance, as well the contribution of anthropogenic environments to the occurrence of the parasites, and give information about the different methods used to collect and identify the nematodes found associated with this species.