Cuban treefrogs, Osteopilus septentrionalis, were grossly examined for parasites and parasite species confirmed by PCR. Angiostrongylus cantonensis larvae were recovered from the hind leg muscle of O. septentrionalis. This is the first report of the zoonotic rat lungworm in the Cuban treefrog and new geographic location (Volusia County) in Florida, US.

Angiostrongylus cantonensis, the rat lungworm (RLW), is a zoonotic nematode parasite that relies on various rat species, mainly Rattus spp., and gastropods to complete its life cycle (Wang et al. 2012). Studies have documented this parasite in gastropod intermediate hosts and rat definitive hosts in several Florida, US, counties from Miami-Dade in the south to Leon in the panhandle (Walden et al. 2020). Additionally, RLW has been reported in a variety of aberrant hosts in Florida, including nonhuman primates and an armadillo, Dasypus novemcinctus (Walden et al. 2020). Paratenic hosts often play an important role in the life cycle of parasites, harboring infective stages of the parasite and possibly facilitating spread to uncommon areas or areas less suitable for intermediate hosts. Several unknowns surround the use of paratenic hosts in the life cycle of A. cantonensis. Ingesting paratenic hosts containing infective, third-stage larvae of RLW, including freshwater prawns/shrimp, land crabs, anurans, lizards, and flatworms has led to human infections in many areas of the world (Wang et al. 2012; Barrett et al. 2016). In Hawaii, invasive Puerto Rican coqui frogs, Eleutherodactylus coqui, and Cuban greenhouse frogs, Eleutherodactylus planirostris, both have had multiple tissue types test positive for RLW by PCR (Niebuhr et al. 2020). These hosts, in conjunction with known intermediate hosts, might contribute to the spread of A. cantonensis and other parasites.

The Cuban treefrog, Osteopilus septentrionalis, is an invasive frog in Florida that is extremely abundant in residential areas in peninsula Florida, often seeking shelter around homes that put them in close contact with humans (Enge 2019). If infected with parasites, the association of O. septentrionalis with humans suggests that they have high potential to serve as agents of zoonotic transmission and might also enable infection of pets consuming them (Cove et al. 2018). Their position in the food webs of Florida makes them well-suited for the life cycle of a variety of parasites, and they are known to eat a variety of snails, insects, and even lizards and frogs (Glorioso et al. 2010). Additionally, their translocation over hundreds of kilometers could spread their parasites to new geographic locations (Enge 2019).

In June 2021, O. septentrionalis adult males (n=13) and females (n=3) were collected in DeLand, Volusia County, in central Florida. The collection site was a stormwater retention ditch in a highly urbanized location. The area houses many businesses with an apartment complex, a convenience store, a dry cleaner, a college campus, a park, and a day care center located within 200 m of the collection site. The animal protocol and sample collection used in this study was approved by the Institutional Animal Care and Use Committee at the University of Florida IACUC #202011222. Frogs were euthanized by immersion in buffered tricaine methanesulfonate (MS-222, Syndel, Ferndale, Washington, USA) and organs and tissues removed and examined for parasites. Sections of the brain, heart, liver, lung, and hind leg skeletal muscle were routinely processed, H&E stained, and microscopically examined for the presence of parasites. The remaining liver, lung, hind leg muscle, and the gastrointestinal tract were examined grossly for parasites. A nematode larva (Fig. 1A) was found in the hind leg muscle of a female O. septentrionalis by microscopic examination. After gross examination of that same frog, two nematode larvae were found encysted in individual muscle fibers (Fig. 1B). Larvae were removed from the muscle tissue and DNA was extracted using the Qiagen DNeasy Blood and Tissue kit per manufacturers protocols (Qiagen, Hilden, Germany). The presence of A. cantonensis was confirmed using conventional, qualitative PCR amplifying a region of the second internal transcribed spacer-2 using primers NC2 and NC5, as previously described (Beveridge and Jabbar 2013). The sequence was deposited in Genbank as accession number MZ825205. No A. cantonensis DNA was present from outside sources during the DNA extraction and PCR processing of this sample, and appropriate assay positive and negative controls were run concurrently.

Figure 1

(A) Photomicrograph of an encysted Angiostrongylus cantonensis larva in the skeletal muscle of a Cuban treefrog (Osteopilus septentrionalis) from Volusia County, central Florida, USA. H&E. 600× magnification. Scale bar=25 µm. (B) Photograph of encysted A. cantonensis larva in the skeletal muscle of the same frog. 80× magnification.

Figure 1

(A) Photomicrograph of an encysted Angiostrongylus cantonensis larva in the skeletal muscle of a Cuban treefrog (Osteopilus septentrionalis) from Volusia County, central Florida, USA. H&E. 600× magnification. Scale bar=25 µm. (B) Photograph of encysted A. cantonensis larva in the skeletal muscle of the same frog. 80× magnification.

Close modal

Although several anuran species have been identified as potential paratenic hosts for A. cantonensis, this is the first record of RLW larvae in the Cuban treefrog and the first record of a frog in Florida serving as a potential paratenic host. Previous reports have identified RLW in Miami-Dade, Orange, Hillsborough, Alachua, St. Johns, and Leon counties in Florida (Walden et al. 2020); our finding confirms its presence in Volusia County. Although the role the Cuban treefrog plays in the life cycle of A. cantonensis remains unknown, larvae in an encysted state in the hind leg muscle suggests it might be a viable paratenic host. Ingestion of infected frogs by accidental hosts could result in incidental infections of wildlife, dogs, and cats, although to date there are no documented reports of this occurrence. There is evidence of cats ingesting O. septentrionalis (Cove et al. 2018). Cuban treefrogs have been implicated in the spread of two nematode species (Aplectana sp. and Oswaldocruzia lenteixeiai) from Cuba to the US (Ortega et al. 2015). Similarly, RLW use of this highly invasive host with a rapidly expanding geographic range might facilitate its movement into new areas of the United States. This finding highlights the necessity for increased awareness of emerging parasites and the role invasive species play in their survival and distribution.

Funding for this work was provided by the University of Florida 2021 Quasi Endowment Grant.

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