Free-living amoebae (FLA) are diverse protozoan organisms that inhabit various environments, including soil, fresh and marine water bodies, and even treated water resources. These amphizoic amoebae can survive independently in the environment and feed on bacteria, algae, and other microorganisms. While they are widely distributed, human infections are relatively uncommon (Brown and De Jonckheere, 1999; Lanocha et al., 2009). However, under certain circumstances, these amoebae can become pathogenic, causing serious infections in humans. Six species have been implicated in human disease: Acanthamoeba spp., Balamuthia mandrillaris, Naegleria fowleri, Vahlkampfia spp., Sappinia pedata, and Vermamoeba vermiformis (Visvesvara, 2010). Amoebae cysts possess a high level of resistance to extreme environmental factors, which plays a crucial role in their ability to survive and spread (Marciano-Cabral and Cabral, 2003).

Some of these amoebic species act as opportunistic parasites, primarily affecting immunocompromised hosts, such as Acanthamoeba spp. and B. mandrillaris, which can lead to infections in the central nervous system (CNS), lungs, sinuses, and skin. In contrast, other species, such as N. fowleri, can impact healthy individuals, leading to severe fulminant acute meningoencephalitis, particularly in children and young adults, in addition to their potential to cause diseases in animals. Acanthamoeba spp. have also been identified as pathogens that can threaten vision, especially in those who wear contact lenses, resulting in keratitis (Schuster and Visvesvara, 2004; Visvesvara et al., 2007). Furthermore, Paravahlkampfia sp. has been identified as a human pathogen that displays characteristic symptoms of primary amoebic meningoencephalitis. (PAM) caused by Naegleria fowleri (Visvesvara et al., 2009). In 2009, a new species belonging to the genus Allovahlkampfia, family Vahlkampfiidae, known as Allovahlkampfia spelaea (A. spelaea), was detected inhabiting caves in Slovenia (Walochnik and Mulec, 2009). To date, the data reported on the possible pathology caused by these amoebae are limited; nevertheless, a recent study identified A. spelaea as a cause of human keratitis in Egypt (Tolba et al., 2016).

The pathogenic potential of FLA is increasing given the circumstances of global climate change, the ecological-human interface, and increased immunosuppressive conditions, with an improvement in the diagnostic capacity for diseased cases. An increase in human interactions with the environment increases the risk of exposure to free-living amoebas, which, in turn, increases the risk of disease (Ozpinar et al., 2020). Raising awareness of FLA disease, fostering heightened clinical suspicion, and making an early diagnosis are essential for effective management and a more favorable outcome for affected individuals. The differential diagnosis of FLA infections is complex because of multiple potential causes, including bacterial, fungal, and other parasitic origins (Kofman and Guarner, 2022).

The pathogenic potential of some species of FLA, whether isolated from the environment or from clinically suspected cases such as Acanthamoeba spp. and N. Fowleri, has been widely investigated in experimental studies (Alves et al., 2016; Feng et al., 2015; Hernández-Jasso et al., 2020; Kim et al., 2008; Marciano-Cabral and Cabral, 2007). Several animal models have been used with different routes of infection, including the intranasal, intraocular, intraperitoneal, and intracranial routes, to elucidate the capacity of environmental isolates of FLA to establish the disease in hosts to investigate its pathogenicity, track disease progression, explore the host immune response toward infection and treat it (Marciano-Cabral and Cabral, 2007).

The pathogenesis and pathophysiology of Allovahlkampfia spp. remain unclear, complicating effective treatment. Ellagic acid (EA), a plant-derived phenolic compound, has multiple biological properties, including antioxidant, anti-inflammatory, anticarcinogenic, antileishmanial, and antimycobacterial activities (Diop et al., 2018; Evtyugin et al., 2020; Maas et al., 1991; Seeram et al., 2008; Srivastava et al., 2007). Similarly, gallic acid shows anti-inflammatory and gastroprotective properties, along with immunomodulatory effects (Evtyugin et al., 2020). The benefits of EA are linked to its capacity to reduce oxidative stress and minimize reactive oxygen species (ROS), which contribute to inflammation and cellular damage (Alfei et al., 2020). These properties indicate that EA and related compounds could serve as potential therapeutic agents for FLA infections, especially for managing oxidative stress and inflammation.

This study aimed to characterize A. spelaea through ultrastructural morphological analysis and investigate its pathogenic potential. Specifically, we focused on elucidating the histopathological changes induced by A. spelaea infection in both the brain and lungs of a murine model. Furthermore, the study included a treatment trial utilizing ellagic acid to assess its potential therapeutic efficacy.