RESEARCH ARTICLE Geographic distribution of Angiostrongylus cantonensis in wild rats (Rattus rattus) and terrestrial snails in Florida, USA Heather D. Stockdale Walden1*, John D. Slapcinsky2, Shannon Roff1, Jorge Mendieta Calle1, Zakia Diaz Goodwin1, Jere Stern1, Rachel Corlett1, Julia Conway1, Antoinette McIntosh1 1 Department of Infectious Disease and Pathology, University of Florida College of Veterinary Medicine, Gainesville, Florida, United States of America, 2 Florida Museum of Natural History, University of Florida, a1111111111 Gainesville, Florida, United States of America a1111111111 a1111111111 * [email protected] a1111111111 a1111111111 Abstract The parasitic nematode Angiostrongylus cantonensis is a major cause of eosinophilic men- OPEN ACCESS ingitis in humans, and has been documented in other incidental hosts such as birds, horses, dogs and non-human primates. It is endemic in Hawaii, and there have been sporadic Citation: Stockdale Walden HD, Slapcinsky JD, Roff S, Mendieta Calle J, Diaz Goodwin Z, Stern J, reports in the southern continental United States. This parasite uses rats as definitive hosts et al. (2017) Geographic distribution of and snails as intermediate hosts. In this study, we collected potential definitive and interme- Angiostrongylus cantonensis in wild rats (Rattus diate hosts throughout Florida to ascertain the geographic distribution in the state: Rats, rattus) and terrestrial snails in Florida, USA. PLoS environmental rat fecal samples, and snails were collected from 18 counties throughout the ONE 12(5): e0177910. https://doi.org/10.1371/ journal.pone.0177910 state. Classical diagnostics and morphological identification, along with molecular tech- niques were used to identify nematode species and confirm the presence of A. cantonensis. Editor: Heike Lutermann, University of Pretoria, SOUTH AFRICA Of the 171 Rattus rattus collected, 39 (22.8%) were positive for A. cantonensis, and 6 of the 37 (16.2%) environmental rat fecal samples collected in three of the surveyed counties Received: January 24, 2017 were also positive for this parasite by real time PCR. We examined 1,437 gastropods, which Accepted: May 5, 2017 represented 32 species; 27 (1.9%) were positive for A. cantonensis from multiple sites Published: May 18, 2017 across Florida. Three non-native gastropod species, Bradybaena similaris, Zachrysia provi- Copyright: © 2017 Stockdale Walden et al. This is soria, and Paropeas achatinaceum, and three native gastropod species, Succinea floridana, an open access article distributed under the terms Ventridens demissus, and Zonitoides arboreus, which are newly recorded intermediate of the Creative Commons Attribution License, hosts for the parasite, were positive for A. cantonensis. This study indicates that A. canto- which permits unrestricted use, distribution, and reproduction in any medium, provided the original nensis is established in Florida through the finding of adult and larval stages in definitive and author and source are credited. intermediate hosts, respectively, throughout the state. The ability for this historically subtrop- Data Availability Statement: All data used to reach ical nematode to thrive in a more temperate climate is alarming, however as the climate the conclusions drawn in this manuscript are found changes and average temperatures rise, gastropod distributions will probably expand, lead- in the manuscript text, tables and figures, and the ing to the spread of this parasite in more temperate areas. Through greater awareness of S1 FASTA file. host species and prevalence of A. cantonensis in the United States, potential accidental Funding: This work was funded by the National infections may be avoided. Center for Veterinary Parasitology (NCVP); www. ncvetp.org. HW received funding for this project in 2013, UF project #00111635. Publication of this article was funded in part by the University of Florida Open Access Publishing Fund. The funders PLOS ONE | https://doi.org/10.1371/journal.pone.0177910 May 18, 2017 1 / 13 Angiostrongylus cantonensis in Florida had no role in study design, data collection and Introduction analysis, decision to publish, or preparation of the manuscript. Angiostrongylus cantonensis, the rat lungworm [1], is a parasitic metastrongyloid nematode that is a major cause of eosinophilic meningitis in humans [2±5], and has been described in Competing interests: The authors have declared over 2800 documented cases in more than 30 countries worldwide [4, 6]. The lifecycle of this that no competing interests exist. parasite has been well described [5, 7±8], and is indirect, requiring multiple hosts for comple- tion. The definitive hosts include several species of rodents, commonly Rattus spp., which ingest the infective third stage larvae (L3) of this parasite found in gastropod intermediate hosts. In some instances, paratenic hosts, such as frogs, crustaceans or fish harbor the infective L3s, and serve as a source of infection, as well [9±13]. Once the L3s are ingested by the rat, they travel passively in the bloodstream before entering the central nervous system and develop to an immature adult. They then re-enter the circulatory system and travel to the pulmonary artery where they become sexually mature. Females lay eggs, which larvate and hatch in the lung tissue, and first stage larvae (L1) are coughed up, swallowed, and shed in feces. Gastro- pods then acquire the L1 that in turn develop to the L3 and continue the cycle. Infections in humans, non-human primates and other animals such as dogs, birds, horses and opossums have been reported [6, 14±21]. These hosts are incidental hosts in which the infective L3s arrest development in the central nervous system. The arrested L3s are unable to develop a patent infection, and subsequent eosinophilic meningitis develops. Both paratenic and incidental hosts become infected through ingesting the infective L3 within the gastropod intermediate host. In humans, adults and children present differently when infected with A. cantonensis. In adults, clinical signs include headache, stiff neck, fever, vomiting and nausea. Paralysis of the face and limbs is also common. In children, nausea, vomiting and fever are more common than headache or stiff neck [4, 6, 22]. In some instances, ocular angiostrongyliasis develops and surgical removal of worms in the eye is necessary [23]. In severe infections, coma or death may occur [24]. Angiostrongylus cantonensis is relatively new to the continental United States, and is pre- sumed to have been introduced in the 1980s from rats arriving on ships into New Orleans, Louisiana [11]. Since its introduction, reported cases of angiostrongyliasis in humans include a young boy who ingested a snail in Louisiana [25] and two young children infected in Texas [26]. However, in Hawaii, where the parasite has been endemic for over 50 years, there have been numerous documented human infections often attributed to intentional or accidental ingestion of snails or slugs [27±29]. In Hawaii, there are several gastropod species reported as potential intermediate hosts of A. cantonensis, including species both native and non-native to the islands [12]. In this recent study [12], over 1200 specimens were examined from collections made throughout the islands, and 6% of non-native gastropods sampled were positive for A. cantonensis larvae. In the continental United States, studies have found snails infected with A. cantonensis in Louisiana and southern Florida [20, 30±32] and infected rats also reported in Louisiana [11, 16]. In Florida, infection with A. cantonensis can be attributed to a non-human primate death in a zoological park and in a privately owned animal, both instances occurring in southern Florida [17, 20]. Other than a few small reports with limited snail species, the extent of the geographic range of A. cantonensis in Florida was unknown. The goal of this study was to survey multiple counties throughout Florida, sampling both definitive and inter- mediate hosts, to obtain a better idea of the geographic range for this zoonotic nematode, and determine whether the gastropod hosts were native or non-native to Florida and the United States. PLOS ONE | https://doi.org/10.1371/journal.pone.0177910 May 18, 2017 2 / 13 Angiostrongylus cantonensis in Florida Methods Ethics statement The animal protocol and sample collection used in this study was approved and performed under the guidelines set forth by the Institutional Animal Care and Use Committee at the Uni- versity of Florida (Protocol #201608110). We obtained permission from property owners before any sample collection. Rat collection and necropsy During 2013±2015, 171 rats (Rattus rattus) were collected from 45 different sites across 6 counties in Florida. In each county, rats were collected from multiple sites. Additionally, 37 environmental rat fecal samples were collected from 24 different sites across 4 counties in Flor- ida. We obtained rats from private facilities through pest control operations that were cur- rently in place, which included bait trapping. Dead rats were collected and stored at -20ÊC until they could be transported to the parasitology laboratory at the University of Florida, and necropsied. The heart, lungs, brain and colon were removed. The left lung lobes and brain were stored in 10% formalin and prepared for histological sectioning. The heart and right lung lobe were stored in 70% ethanol and examined for the presence of adult Angiostrongylus canto- nensis. Any adult nematodes were identified based on