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.