Older urban rats are infected with the zoonotic nematode Angiostrongylus cantonensis.

Rats, being synanthropic, are hosts to agents of zoonotic diseases that pose a threat to human and domestic animal health. The nematode parasite Angiostrongylus cantonensis, commonly known as the rat lungworm, is no exception; it can cause potentially fatal neural disease in humans, dogs and other species. The distribution of A. cantonensis (haplotypes SYD.1 and Ac13) and its close relative, Angiostrongylus mackerrasae is not well understood in Australia. We investigated the prevalence of Angiostrongylus in rats in Sydney, Australia, primarily via faecal qPCR, and identified the species and haplotypes using partial cox1 sequencing. We found a moderate prevalence of infection (29%; 95% CI: 16.1-46.6%) in black (Rattus rattus) and brown (Rattus norvegicus) rats around public parks and residential areas. This study demonstrates that Sydney's urban rat population is a reservoir for A. cantonensis. Modelling infection status as a function of rat species, sex, tibia length (as a proxy for age), and health index (a measure of weight by size) revealed that older rats are statistically more likely to be infected (χ 2 1 = 5.331, P = 0.021). We observed a dominant presence of the A. cantonensis SYD.1 haplotype, for which the implications are not yet known. No A. mackerassae was detected, leading us to suspect it may have a more restricted host- and geographical range. Overall, this study illustrates the presence and potential risk of A. cantonensis infection in Sydney. Public education regarding transmission routes and preventative measures is crucial to safeguard human and animal health.

Fatal neural angiostrongyliasis in the Bolivian squirrel monkey (Saimiri boliviensis boliviensis) leading to defining Angiostrongylus cantonensis risk map at a zoo in Australia.

Neural angiostrongyliasis (NA) is a parasitic disease caused by Angiostrongylus cantonensis (rat lungworm). This study presents a case of NA in a captive Bolivian squirrel monkey from a zoo in western Sydney, Australia. The objective was to identify the A. cantonensis cox1 haplotype responsible for the infection and compare its mitochondrial DNA (mtDNA) to known Australian mtDNA. An epidemiological investigation was conducted to assess the risk of infection, focusing on the resident rat population in the zoo. Methods involved trapping rats and collecting rat faeces for Angiostrongylus detection, speciation, and cox1 haplotype confirmation. Various techniques were employed, including necropsy, morphological examination, and molecular methods such as ITS-2 qPCR, cox1 sequencing, and ITS-2 metabarcoding. Cluster analysis of rat faeces distribution and Angiostrongylus detection utilised an equal sampling effort (ESE) approach. Gastropods were collected throughout the study for Angiostrongylus surveillance using a hypersensitive qPCR assay. Results revealed significant clustering of rat faeces near exhibits with fresh food provision and absence of predators. Angiostrongylus-positive faeces were uniformly distributed across the zoo property. Mitochondrial DNA analysis confirmed the presence of the Ac13 haplotype of A. cantonensis in the monkey. Morphology, ITS-2 metabarcoding and partial cox1 sequencing detected only A. cantonensis, with the Ac13 cox1 haplotype predominating. A high prevalence of infection (64%, 9/14) was found in brown rats, with quantification of larvae indicating high shedding rates. Co-infections with both Ac13 and local SYD.1 A. cantonensis cox1 haplotypes were observed. Only three gastropods (all of which were Angiostrongylus-negative) were found in the survey. To minimise the risk of exposure for susceptible species, targeted rodent control was implemented in areas with higher exposure risk. A potential strategy (which requires further exploration) to consider for future zoo design was suggested. This study provides insights into the epidemiology and genetic diversity of A. cantonensis in Australia, emphasising the importance of control measures to prevent future outbreaks.

Angie-LAMP for diagnosis of human eosinophilic meningitis using dog as proxy: A LAMP assay for Angiostrongylus cantonensis DNA in cerebrospinal fluid.

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.

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.

What the fox? Cryptic Eucoleus [Capillaria] sp. in the respiratory tract of a cat from Australia.

Eucoleus aerophilus (syn. Capillaria aerophila) is a zoonotic trichuroid nematode parasite of dogs, cats and wild carnivores with a global distribution. The main reservoir species in Europe is the red fox, where it has been detected in up to 97% of animals surveyed. Despite the burgeoning feral cat and fox population in Australia, there is a paucity of information about the occurrence and molecular identity of E. aerophilus in these species. The occurrence of a gravid capillariid nematode in the bronchoalveolar lavage of a 12-week-old kitten from central New South Wales (NSW), with a history of lower respiratory signs that had been non-responsive to treatment with metronidazole or amoxicillin-clavulanic acid, prompted a detailed morphological and molecular investigation into the identity of the parasite including the examination of opportunistically-collected red fox tracheas from the region. A combination of PCR and next-generation sequencing yielded the first complete mitochondrial genome of E. aerophilus, collected from the red foxes in Australia, and revealed the presence of a cryptic Eucoleus [Capillaria] sp. in the kitten from central NSW. The protein-coding genes were 14-23% and 5-30% different (pairwise distance) at the nucleotide and amino acid sequences, respectively, which suggests the occurrence of a genetically distinct Eucoleus sp. lineage in Australia. The phylogenetic analysis using both Bayesian and the maximum likelihood methods demonstrated monophyly of the Trichuridae plus Capillariidae using amino acid sequences encoded by mitochondrial DNA. Analysis based on complete SSU rDNA sequences of Eucoleus [Capillaria] sp. and E. aerophilus placed them within Eucoleus spp. from the respiratory tract of their hosts. While Eucoleus spp. may not currently pose a significant threat to companion animals in Australia, their status as a recently emerged pathogen in Europe suggest that greater efforts should be made to understand the distribution and epidemiology of these parasites.

Apparent lack of spill-over of parasites from an invasive anuran: PCR detects Entamoeba in cane toads (Rhinella marina) but not in sympatric Australian native frogs.

The recent detection of a novel amoebozoan parasite (Entamoeba sp. CT1) killing invasive cane toads (Rhinella marina) in tropical Australia raises concerns of potential spill-over into native anuran populations. Considering the vulnerability of anuran communities globally, Entamoeba sp. CT1 may pose a serious threat to anuran biodiversity. Through PCR-based detection and molecular identification, we investigated the prevalence of Entamoeba spp. in the faeces and colon tissue of cane toads (Rhinella marina) and eleven native Australian frog species from a single locality in the Northern Territory. No Entamoeba DNA was detected in samples of native frog faeces (N = 57) or colons (N = 17). Entamoeba DNA was detected in 24% of 45 cane toads (95%CI 14.08-38.82). Both E. ranarum and Entamoeba sp. CT1 were present in cane toads. The failure of faecal samples to indicate Entamoeba spp. in infected cane toads may be due to cysts in faeces being shed intermittently, degraded before analysis, or impervious to lysis prior to DNA isolation. Our results suggest that native frogs do not carry the pathogen in an area where 20-30% of cane toads are infected with Entamoeba sp. CT1. We demonstrate the importance of recognising PCR inhibition prior to molecular diagnostics, and the apparent inadequacy of faecal samples for the detection of Entamoeba spp. in anurans.