Most cited article - PubMed ID 33252651
AcanR3990 qPCR: A Novel, Highly Sensitive, Bioinformatically-Informed Assay to Detect Angiostrongylus cantonensis Infections
Tenrecs (Afrosoricida: Tenrecidae) are insectivorous mammals endemic to Madagascar, currently facing population declines due to habitat loss and subsistence hunting. Emerging infectious diseases, including parasitic infections, may pose additional threats. A comparable situation has been observed in Algerian hedgehogs (Atelerix algirus) in Mallorca, where the invasive nematode Angiostrongylus cantonensis has been associated with severe neuropathology. Given ecological parallels and the confirmed presence of A. cantonensis in Madagascar, this study aimed to assess its potential impact on tenrec health and survival. An experimental infection was conducted using Echinops telfairi, orally inoculated with 500 or 2000 third-stage larvae and monitored for 59 days through behavioral observations. Following euthanasia, artificial tissue digestion, qPCR analysis, and histopathology were performed. Baermann's larvoscopy was used to examine feces from day 39 post-infection (DPI). No neuropathological symptoms were observed. Artificial digestion revealed 11 third-stage larvae in the gastrointestinal tract of one high-dose tenrec at 36 DPI. Parasite DNA was detected in various organs of both groups; however, accumulation in brain tissue occurred only in the high-dose group, with no viable larvae visible in histological sections. E. telfairi showed no apparent sensitivity to A. cantonensis, unlike the Algerian hedgehog, which develops severe neuropathology under comparable conditions. These findings suggest that A. cantonensis exhibits host-specific outcomes, and not all mammals act as aberrant hosts facing fatal infection. The persistence of third-stage larvae up to 36 DPI raises concerns about potential transmission to predators or hypothetical human infection.
- Keywords
- Angiostrongylus cantonensis, Echinops telfairi, Invasion, Madagascar,
- MeSH
- Angiostrongylus cantonensis * physiology MeSH
- Feces parasitology MeSH
- Strongylida Infections * veterinary parasitology pathology MeSH
- Larva physiology MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
- Geographicals
- Madagascar epidemiology MeSH
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.
- Keywords
- Angiostrongylus cantonensis, intermediesis, paratenic host, rat lungworm, reptiles,
- MeSH
- Angiostrongylus cantonensis * physiology MeSH
- Snakes parasitology MeSH
- Strongylida Infections * parasitology transmission veterinary MeSH
- Lizards * parasitology MeSH
- Rats MeSH
- Larva physiology MeSH
- Rats, Wistar MeSH
- Food Chain * MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Animals MeSH
- Publication type
- Journal Article 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.
- Keywords
- Angiostrongylus cantonensis, caudal autotomy, lizards, rat lungworm, sentinels,
- MeSH
- Angiostrongylus cantonensis * isolation & purification MeSH
- Strongylida Infections * epidemiology veterinary parasitology MeSH
- Lizards * parasitology MeSH
- Prevalence MeSH
- Sentinel Species parasitology MeSH
- Animals MeSH
- Check Tag
- Male MeSH
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Geographicals
- Spain epidemiology 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.
- Keywords
- Angiostrongylus cantonensis, Gastropods, Larvae release, Stress stimuli,
- MeSH
- Angiostrongylus cantonensis * physiology MeSH
- Stress, Physiological * MeSH
- Mucus MeSH
- Strongylida Infections parasitology MeSH
- Real-Time Polymerase Chain Reaction MeSH
- Larva * physiology MeSH
- Gastropoda parasitology MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
Human angiostrongylosis is an emerging zoonosis caused by the larvae of three species of metastrongyloid nematodes of the genus Angiostrongylus, with Angiostrongylus cantonensis (Chen, 1935) being dominant across the world. Its obligatory heteroxenous life cycle includes rats as definitive hosts, mollusks as intermediate hosts, and amphibians and reptiles as paratenic hosts. In humans, the infection manifests as Angiostrongylus eosinophilic meningitis (AEM) or ocular form. Since there is no comprehensive study on the disease in the Indian subcontinent, our study aims at the growing incidence of angiostrongylosis in humans, alongside its clinical course and possible causes. A systematic literature search revealed 28 reports of 45 human cases from 1966 to 2022; eosinophilic meningitis accounted for 33 cases (75.5%), 12 cases were reported as ocular, 1 case was combined, and 1 case was unspecified. The presumed source of infection was reported in 5 cases only. Importantly, 22 AEM patients reported a history of eating raw monitor lizard (Varanus spp.) tissues in the past. As apex predators, monitor lizards accumulate high numbers of L3 responsible for acute illness in humans. For ocular cases, the source was not identified. Most cases were diagnosed based on nematode findings and clinical pathology (primarily eosinophilia in the cerebrospinal fluid). Only two cases were confirmed to be A. cantonensis, one by immunoblot and the other by q-PCR. Cases of angiostrongylosis have been reported in Delhi, Karnataka, Kerala, Maharashtra, Madhya Pradesh, Puducherry, Telangana, and West Bengal. With a population of more than 1.4 billion, India is one of the least studied areas for A. cantonensis. It is likely that many cases remain undetected/unreported. Since most cases have been reported from the state of Kerala, further research may focus on this region. Gastropods, amphibians, and reptiles are commonly consumed in India; however, typical preparation methods involve cooking, which kills the nematode larvae. In addition to studying rodent and mollusk hosts, monitor lizards can be used as effective sentinels. Sequence data are urgently needed to answer the question of the identity of Angiostrongylus-like metastrongylid nematodes isolated from all types of hosts. DNA-based diagnostic methods such as q-PCR and LAMP should be included in clinical diagnosis of suspected cases and in studies of genetic diversity and species identity of nematodes tentatively identified as A. cantonensis.
- Keywords
- Angiostrongylus cantonensis, Indian subcontinent, eosinophilic meningitis, human angiostrongyliasis,
- Publication type
- Journal Article MeSH
- Review 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 * genetics MeSH
- Angiostrongylus * genetics MeSH
- Molecular Diagnostic Techniques MeSH
- Snails genetics MeSH
- Strongylida Infections * diagnosis veterinary MeSH
- Rats MeSH
- Humans MeSH
- Meningitis * diagnosis veterinary MeSH
- Dogs MeSH
- DNA, Ribosomal MeSH
- Nucleic Acid Amplification Techniques MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Humans MeSH
- Dogs MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- DNA, Ribosomal MeSH
Angiostrongylus cantonensis is an invasive zoonotic nematode which causes eosinophilic meningitis in accidental hosts – vertebrates including humans – and is known to impact wildlife. Even though the parasite originates in Southeast Asia, it has spread worldwide, especially into fragile island ecosystems. The Canary Islands are a biodiversity hot spot with numerous endemic species affected by biological invasions. Among others, Rattus rattus and Rattus norvegicus threaten many endemic species by predation and may spread invasive pathogens, such as the rat lungworm A. cantonensis, which was first described in Tenerife in 2010. Since it is known that lizards can act as paratenic hosts for A. cantonensis and Gallotia galloti is a lizard abundant in Tenerife, the aim of this study was to reveal the role of these endemic lizards in the parasite's life cycle. Gallotia galloti were captured in Tegueste, Tenerife, and after euthanasia, liver and tail muscle samples were examined for the presence of A. cantonensis. During microscopic examination, 8/36 liver samples (22.2%) contained granulomas with nematode larvae. In total, 10/39 liver samples (25.6%) and 7/36 tail muscle samples (19.4%) were positive for A. cantonensis DNA using qPCR. This is a first report of a reptile endemic to the Canary Islands acting as paratenic host of A. cantonensis. The fact that the parasite is obviously well-established in the island ecosystem and exploits endemic lizards as hosts may have important implications for the parasite's ecoepidemiology. Moreover, the parasite might threaten other species which depend on lizards in the island trophic web.
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- Publication type
- Journal Article MeSH