Figure 1

Figure 1. Clinical manifestation of cutaneous pythiosis in 2 dogs, Italy. A, B) Case 1, showing a large mass on the right flank (A) and a large mass on the left axilla...

We evaluated soft tissue swellings in 2 unrelated dogs that lived 30 km apart in Rome province of central Italy in October 2022 (case 1) and January 2023 (case 2) (Figure 1). The animals had never been outside of Italy before onset of skin lesions and were otherwise healthy. We collected anamnesis and clinical manifestation data (Table).

Figure 2

Figure 2. Histological analyses of cutaneous pythiosis in 2 dogs, Italy. A) Section from punch biopsy of cutaneous mass showing confluent pyogranulomatous and eosinophilic granulomas with central necrosis in the deep dermis...

Histopathologic results of lesion punch biopsies showed multifocal to coalescing (pyo)granulomatous and eosinophilic dermatitis and panniculitis with intralesional, irregular branching hyphae (Figure 2, panel A). The hyphae were positive for Grocott methenamine silver stain, appearing dark brown (Figure 2, panel B), whereas they were negative after staining with periodic acid Schiff stain. Culturing on Sabouraud dextrose agar (for fungi) containing chloramphenicol and gentamycin yielded negative results.

To obtain a definitive identification, we extracted genomic DNA from biopsied tissues by using a NucleoSpin Tissue kit (Macherey-Nagel, https://www.mn-net.com). We PCR amplified and sequenced the internal transcribed spacer (ITS) region by using the primer pair ITS4 and ITS5 (11). We obtained identical sequences (GenBank accession nos. OQ532907 [case 1] and OQ532908 [case 2]) that were closely related to ITS sequences of P. insidiosum from GenBank (compared by using BLASTn, https://blast.ncbi.nlm.nih.gov). We aligned sequences belonging to different clusters within the P. insidiosum complex (5) by using MEGA11 software (https://www.megasoftware.net). We conducted phylogenetic analysis by using the neighbor-joining method (bootstrap analysis with 1,000 replicates); sequences obtained from the infected dog tissues clustered with Pythium cluster IV (Appendix Figure), corresponding to the newly described species P. periculosum (5).

Although an environmental study would be necessary for confirmation, Lake Bracciano near Rome might have been the infection source (Table). Both dogs regularly swam in the lake, which has vast stagnant waters with stable temperatures of ≈30°C in hotter months, features known to support pathogenic Pythium spp. growth (2,3).

Additional material was collected from the lesion in case 2 through fine-needle aspiration. We suspected the organism failed to grow in previous cultures because of chloramphenicol supplementation (10). Therefore, we cultured the aspirate on unsupplemented Sabouraud dextrose agar at 37°C. After a 24-hour incubation, submerged, colorless colonies with irregular radiate patterns developed from the aspirated material (Figure 2, panel C). Microscopically, the hyphae were broad (4–10 µm in diameter), hyaline, and sparsely septate (Figure 2, panel D). Colonies were identified as P. periculosum by using the same molecular approach applied to biopsies.

Our cases suggest a geographic distribution of pythiosis broader than previously recognized, aligning with other reports of the disease outside of classical tropical and subtropical areas, such as US regions near the border of Canada, as well as Spain, Israel, Japan, and South Korea (2,3). Because the average climatic conditions in temperate zones appear unsuitable for the causative oomycete life cycle, cases in those areas likely occur in restricted ecologic niches. Our report illustrates that concept; central Italy has a primarily Mediterranean climate that has mild, sometimes rainy winters and sunny, hot, and usually dry summers. Those features do not fit the description of areas more prone to pythiosis. However, the zone where we suspect the dogs became infected has features of a pythiosis-risk area (warm, humid environment). Increasing reports of pythiosis in nonendemic regions might indicate an expansion of the causative oomycetes because of global climate changes. Another interpretation is that more cases are recognized and published because of increased awareness of healthcare personnel and availability of diagnostic tools (3). Environmental and clinical studies will be necessary to address those hypotheses.

The dogs in this report showed signs typically associated with the cutaneous form of pythiosis, including developing multiple masses or dermal plaques over time (3). Gastrointestinal pythiosis in dogs is another form of the disease characterized by weight loss, vomiting, diarrhea, and hematochezia (3,12).

Genetic variation associated with geographic provenance exists for species within the P. insidiosum complex (4,5). P. insidiosum cluster I is mainly found throughout the Americas, whereas P. insidiosum cluster II is typically found in Asia and Australia. P. periculosum cluster III has been reported mainly in the United States and is sympatric with members of cluster I (5). The genotype identified in our cases (P. periculosum cluster IV) seems to have a broader distribution; most reports of human cases have been from Thailand and India (3). However, P. periculosum cluster IV has also been recovered from environmental samples in Brazil and the United States (13,14) and from human patients in Israel (15) and Spain (10), which is noteworthy because of the close proximity of Spain to Italy.

In conclusion, from a One Health perspective, our study shows the environmental presence of an unexpected, exotic pathogen that could potentially infect humans in a country with a temperate climate. In Italy, pythiosis should now be considered a differential diagnosis by human and veterinary health professionals, especially in cases where there is a history of exposure to freshwater habitats.