Figure 1

Figure 1. Sampling locations used for detection of rat lungworm (Angiostrongylus cantonensis) in rats and gastropods, Italy. Solid black circles indicate sites from which A. cantonensis–positive samples were...

We obtained a total of 32 frozen rat specimens, 10 R. rattus and 22 R. norvegicus, from a pest control company operating in metropolitan Naples and its surroundings. We conducted an initial sampling phase randomly across various locations. After we detected A. cantonensis lungworm in rats, we conducted a second sampling, during which we collected 352 gastropods from locations where infected rats were collected and from nearby areas selected at random. In total, we sampled rodent and gastropod samples from 15 locations during January–November 2024 (Figure 1; Appendix 1).

We necropsied rats at the Experimental Zooprophylactic Institute of Southern Italy in Naples. We isolated the heart and lungs and inspected them for adult Angiostrongylus spp. worms, characterized by the distinctive barber pole appearance in female nematodes; we preserved isolated worms in 96% ethanol. We froze tissue samples from the brain, heart, kidneys, liver, lungs, and spleen of rats for molecular analysis. We confirmed rat species on the basis of DNA extracted from spleen samples by using the Nucleic Acid Extraction Kit (Magnetic Bead Method) (Zybio, https://www.zybio.com), followed by amplification of the mitochondrial cytochrome b gene (Appendix 2). We detected A. cantonensis nematodes in 13 (40.6%) of 32 rats collected from 3 locations, with a mean of 7 (range 1–24) worms per rat (Table; Figure 1; Appendix 1).

Figure 2

Figure 2. Maximum-likelihood phylogenetic tree of rat lungworm (Angiostrongylus cantonensis) detected in rats and gastropods, Italy. Tree is based on cytochrome c oxidase subunit 1 gene (1,578 bp) and partial...

Molecular analysis confirmed A. cantonensis nematodes in 12 positive rats, and we subsequently sequenced 69 adult worms. We extracted DNA from those adult worms by using the DNeasy Blood & Tissue Kit (QIAGEN, https://www.qiagen.com), and we amplified the complete cytochrome c oxidase subunit 1 (CO1) gene (Appendix 2). The obtained sequences revealed 2 distinct haplotypes (NAP1 and NAP2), differing by 2 single-nucleotide polymorphisms (SNPs) at positions 1092 and 1481 of the CO1 gene; the SNP at position 1481 resulted in a different amino acid. In a maximum-likelihood phylogenetic tree (Figure 2), both haplotypes clustered within clade II sensu, as previously defined (9), alongside other sequences from Europe, except for 1 (GenBank accession no. PP468354; 215 bp) from Valencia that clustered in a separate clade, a sister to clade I, differing from all other sequences from Europe in 3 SNPs. Compared with the TEN.1 isolate (GenBank accession no. MK570629) from Tenerife, Spain, each haplotype from Italy differed by a single SNP: NAP1, detected in Naples (Porto) and Marano di Napoli, differed at position 1092; and NAP2, detected in Naples (Vomero), differed at position 1481. The 394-bp sequence from Mallorca, Spain (GenBank accession no. MN227185), was identical to NAP2. Among the Valencia isolates, 10 sequences were identical to NAP1, 3 were identical to NAP2, and the rest differed in 1, 2, or 3 SNPs from the other sequences from Italy.

Collected gastropods were identified to species level by a trained malacologist on the basis of morphological criteria. Molecular identification was performed on juvenile and shell-less specimens lacking distinct morphological characteristics. DNA was extracted from muscle tissue using the same protocol used for rat spleen samples, with an extended overnight prelysis phase at 56°C, optimized for the L3 stage of A. cantonensis. Molecular identification was made on the basis of sequences of the mitochondrial 16S rRNA gene (Appendix 2). We detected A. cantonensis worms by using a species-specific quantitative PCR on DNA isolated from gastropod tissue (11). Of the 352 gastropods examined, 8 (2.3%) gastropods from 2 localities tested positive for A. cantonensis DNA (Figure 1, Table; Appendix 2). We successfully obtained 6 CO1 gene fragment sequences from the 8 positive gastropods (Appendix 2), and compared those with sequences from adult A. cantonensis lungworms from rats in this study. All sequences belonged to the NAP1 haplotype.

We provide robust evidence that the A. cantonensis rat lungworm is in the central Mediterranean region in the Naples area of Italy. Circulation of this zoonotic nematode in the highly populated Naples metropolitan area is concerning because of its ability to cause severe neurologic and ocular disorders in humans. Because Naples has an environment ideal for A. cantonensis transmission to the human population, enhanced awareness is needed among healthcare practitioners and diagnostic protocols should be revised and applied locally in the differential diagnosis of meningoencephalitis cases (12). In addition, considering reported clinical cases in domestic animals and in wildlife in known endemic foci (13–15), veterinary practitioners in the Naples area should be alerted.