Background/Objectives: Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, remains a major neglected tropical disease, with over six million cases concentrated, primarily in Latin America. Despite decades of research, treatment continues to rely on two outdated drugs—benznidazole and nifurtimox—both of which exhibit limited efficacy and are associated with severe side effects. In this context, drug repurposing presents a promising strategy to accelerate the development of safer and more effective therapies. Nitroxoline, a hydroxyquinoline compound widely used in Europe to treat bacterial urinary tract infections, has recently garnered attention for its broad-spectrum antimicrobial and anticancer activities. This study evaluated the antitrypanosomal potential of nitroxoline against both epimastigote and intracellular amastigote forms of T. cruzi, demonstrating significantly greater efficacy than benznidazole. Methods: In addition to its antiparasitic activity, we investigated the mechanism of parasite death and found that nitroxoline induces hallmarks of programmed cell death, including chromatin condensation, mitochondrial membrane depolarization, ATP depletion, reactive oxygen species accumulation, and increased membrane permeability. These cellular events are critical for minimizing host tissue inflammation and suggest a safer therapeutic profile. Results: The nitroxoline was shown to induce greater activity than the reference treatment, benznidazole, in addition to triggering events related to apoptotic or silent cell death. Conclusions: Given its established clinical use and favorable safety data, nitroxoline emerges as a strong candidate for further investigation as a repurposed treatment for Chagas disease. Future work should focus on in vivo efficacy, pharmacokinetics, and drug delivery strategies to enhance systemic bioavailability.

Contrawise to mosquitoes, there is no country-wide long-term surveillance of ticks in Germany, leading to large gaps in coverage for distribution data. Here we report on results from two consecutive tick studies in northern Rhineland-Palatinate, western Germany, conducted in 2022 and 2023. In 2022, a new focus of the ornate sheep tick Dermacentor marginatus was detected at a military training area east of Coblenz („Schmidtenhöhe“) which is partially managed as a nature reserve where old races of cattle and horses are grazing. Two unexpected further foci were discovered north and west of the city in 2024. In 2023, repeated flagging in a small area of a forest southwest of Coblenz (“Stadtwald”) revealed four nymphs of the passerine bird tick Ixodes frontalis. However, over 99

Phlebotomine sand flies of the genus Sergentomyia are considered to be of minor importance as vectors of Leishmania parasites pathogenic to humans, but are known to transmit lizard parasites of the subgenus Sau-roleishmania, including L. (S.) adleri. However, knowledge on the geographic distribution of Sauroleishmania spp. and the infection rates in the vectors is very limited. Therefore, our study aimed (1) to further elucidate the distribution and prevalence of Sauroleishmania spp. in their respective vectors and (2) to assess the potential risk for occasional transmission of Leishmania parasites to international military personnel deployed in camps in Mali and Niger. A total of 1,482 wild-caught sand flies (Sergentomyia spp. and closely related Grassomyia spp.) were screened by real-time PCR for the presence of Leishmania DNA. Thirty-two sand fly pools were tested positive, with six from Mali and 26 from Niger. The DNA of four representative isolates was sequenced. The resulting sequences revealed a homology to L. adleri, which leads to the first report of this species from Mali and Niger to the best of our knowledge. The results suggest that Sergentomyia (Sintonius) clydei might be the natural sand fly vector, while Grassomyia spp. appear to be refractory. No Leishmania sp. pathogenic to humans was detected in these sand flies.

Acanthamoeba is a ubiquitous genus of amoebae that can act as opportunistic parasites in both humans and animals, causing a variety of ocular, nervous and dermal pathologies. Despite advances in Acanthamoeba therapy, the management of patients with Acanthamoeba infections remains a challenge for health services. Therefore, there is a need to search for new active substances against Acanthamoebae. In the present study, we evaluated the amoebicidal activity of nitroxoline against the trophozoite and cyst stages of six different strains of Acanthamoeba. The strain A. griffini showed the lowest IC50 value in the trophozoite stage (0.69 ± 0.01 µM), while the strain A. castellanii L-10 showed the lowest IC50 value in the cyst stage (0.11 ± 0.03 µM). In addition, nitroxoline induced in treated trophozoites of A. culbertsoni features compatibles with apoptosis and autophagy pathways, including chromatin condensation, mitochondrial malfunction, oxidative stress, changes in cell permeability and the formation of autophagic vacuoles. Furthermore, proteomic analysis of the effect of nitroxoline on trophozoites revealed that this antibiotic induced the overexpression and the downregulation of proteins involved in the apoptotic process and in metabolic and biosynthesis pathways.

Among the pathogenic free-living amoebae (FLA), Naegleria fowleri is the etiological agent of a fatal disease known as primary amoebic meningoencephalitis (PAM). Once infection begins, the lesions generated in the central nervous system (CNS) result in the onset of symptoms leading to death in a short period of time. Currently, there is no standardized treatment against the infection, which, due to the high virulence of the parasite, results in a high case fatality rate (>97%). Therefore, it is essential to search for new therapeutic sources that can generate a rapid elimination of the parasite. In recent years, there have already been several successful examples of drug repurposing, such as Nitroxoline, for which, in addition to its known bioactive properties, anti-Balamuthia activity has recently been described. Following this approach, the anti-Naegleria activity of Nitroxoline was tested. Nitroxoline displayed low micromolar activity against two different strains of N. fowleri trophozoites (IC50 values of 1.63 ± 0.37 µM and 1.17 ± 0.21 µM) and against cyst stages (IC50 of 1.26 ± 0.42 μM). The potent anti-parasitic activity compared to the toxicity produced (selectivity index of 3.78 and 5.25, respectively) in murine macrophages and human cell lines (reported in previous studies), together with the induction of programmed cell death (PCD)-related events in N. fowleri make Nitroxoline a great candidate for an alternative PAM treatment.

To perform PCR from serum for the diagnosis of visceral leishmaniasis is convenient and much less invasive than the examination of deeper compartments such as bone marrow. We compared three Leishmania-specific real-time PCRs with three different molecular targets (kinetoplast DNA, the small subunit-ribosomal RNA-(ssrRNA-)gene, the glucose-6-phosphate isomerase-(gpi-)gene) regarding their sensitivity and specificity in human serum. Residual sera from previous diagnostic assessments at the German National Reference Center for Tropical Pathogens Bernhard Nocht Institute for Tropical Medicine Hamburg and the Swiss Tropical and Public Health Institute were used. The sensitivities of kinetoplast DNA-PCR, ssrRNA-gene PCR, and gpi-PCR were 93.3%, 73.3%, and 33.3%, respectively, with 15 initial serum samples from visceral leishmaniasis patients, as well as 9.1%, 9.1%, and 0.0%, respectively, with 11 follow-up serum samples taken at various time points following anti-leishmanial therapy. Specificity was 100.0% in all assays as recorded with 1.137 serum samples from deployed soldiers and migrants without clinical suspicion of visceral leishmaniasis. Kinetoplast-DNA PCR from serum was confirmed as a sensitive and specific approach for the diagnosis of visceral leishmaniasis. The results also indicate the suitability of serum PCR for diagnostic follow-up after therapy, in particular regarding therapeutic failure in case of persisting positive PCR results.

Most of our knowledge on the processes structuring parasite communities in amphibians originate from temperate-zone taxa, whereas Afrotropical communities have been neglected so far. We found evidence that ecological fitting of the hosts and, probably, differential immune response may influence the variation in parasite species richness, prevalence, and infestation intensity of East African frogs Hyperolius kivuensis and H. viridiflavus. The most closely related host species share the same macrohabitat (that implies the same pool of potential parasites), but differ in microhabitat preference, so that a comparative analyses of syntopic and allopatric populations is expedient to reveal ecological fitting. We detected 11 parasite species (one annelid, four nematodes, five trematodes, one cestode) and two endocommensal species (protozoans). The component parasite communities included 4–5 helminth species in H. kivuensis and 6–8 in the more aquatic H. viridiflavus, supporting the hypothesis that trematode diversity increases with the amount of time spent in water. Five parasite species (Orneoascaris chrysanthemoides, Clinostomum chabaudi, an undetermined echinostomatid) and two protozoans (Nyctotheroides sp., and Protoopalina sp.) are shared among the syntopic amphibian populations. This finding indicates a similar susceptibility of these amphibians to infestation from the local parasite pool. Yet, the low prevalence of single- and multi-species infestations in H. kivuensis indicates that parasite clearing by its immune response is probably more effective and prominent than in H. viridiflavus. Therefore, H. viridiflavus suffered from significantly reduced short-term survival due to the infection. Thus, we conclude that the processes structuring component parasite communities in amphibians do not differ generally between temperate-zone and Afrotropical host species, but they do in the magnitude of ecological fitting.

Clinostomum metacercariae are common endoparasites of fish and frogs. In this study, we examine taxonomic identity and developmental strategy of Clinostomum metacercariae infesting reed frogs Hyperolius kivuensis and H. viridiflavus in Rwanda. Moreover, we evaluate the impact of infestation on demographic and morphological life-history traits of the hosts. Morphological and molecular features, particularly genital morphology and COX1 sequences, provided evidence that the metacercariae belong to C. chabaudi Vercammen-Grandjean, 1960. Depending on the host’s defensive behavior and the availability of resources, metacercariae develop either as sedentary “yellow grubs” encysted in the lymphatic sacs or mouth of the host or as initially encysted, but later free-ranging individuals invading the host´s body cavity. Nutrition on lymphatic fluid within the cyst leads to yellow-colored gut content, feeding on blood or host tissue, to brownish green gut content in free-ranging individuals. Almost all metacercariae opted for the first developmental strategy in H. kivuensis, whereas the second strategy dominated in metacercariae infesting H. viridiflavus. Hyperolius kivuensis suffered significant morphological modifications, when infested with encysted metacercariae. Both developmental modes permitted a coexistence with the host of less than one year. We hypothesize that the presence of alternative development modes is an adaptation of C. chabaudi to cope with resource limitation within host-produced cysts.

Species of MegalobatrachonemaYamaguti, 1941 (Ascaridida: Cosmocercoidea) are important nematode parasites in amphibians and reptiles. However, the phylogenetic relationship of its included two subgenera Megalobatrachonema and Chabaudgolvania remains unclear. In the present study, a new species of Megalobatrachonema, M. (Chabaudgolvania) wangi sp. nov., was described based on the specimens collected from the lesser spiny frog Quasipaa exilispinosa (Liu & Hu) (Amphibia: Anura) in China. The ribosomal [large ribosomal DNA (28S) and internal transcribed spacer (ITS1-5.8S-ITS2)] and mitochondrial [12S small subunit ribosomal DNA and cytochrome c oxidase subunit 1 (cox1)] target regions of the new species and M. (Chabaudgolvania) terdentatum, together with the 12S region of M. (Megalobatrachonema) hainanensis, were amplified and sequenced for molecular identification and phylogeny. Moreover, in order to clarify the systematic position of the new species and the phylogenetic relationship of the two subgenera Megalobatrachonema and Chabaudgolvania, phylogenetic analyses based on 28S + ITS1-5.8S-ITS2 + 12S sequence data were performed using maximum likelihood (ML) inference and Bayesian inference (BI). The molecular phylogenetic results conflicted with the current classification and challenged the validity of the subgenus Chabaudgolvania, that should be a synonym of the subgenus Megalobatrachonema. The presence or absence of valves in the oesophageal bulb as a key criterion for delimitation of the two subgenera Megalobatrachonema and Chabaudgolvania seems to be unreliable.

The diversity of nematodes infecting amphibians is understudied in tropical Africa and unknown in Rwanda. Diversity assessment is hampered by the fact that species descriptions refer mostly to morphological features that are unlinked to DNA sequences of marker genes available in public databases. In this paper, we explore the abundance and diversity of parasitic nematodes in reed frogsHyperolius kivuensis(n= 115),H. parallelus(n= 45) andH. viridiflavus(n= 100) collected in Rwanda. Five nematode species were identified morphologically asOrneoascaris chrysanthemoides, O. schoutedeni, Gendria leberrei, Aplectana chamaeleonisandRhabdias collaris. Corresponding DNA sequences of 18S and COI genes were determined and subsequently deposited in GenBank.Aplectana chamaeleonisshowed the highest prevalence (8.7%), butO. chrysanthemoidesthe highest mean intensity of infection (6.0) and largest number (24) of individuals inH. kivuensis. To the best of our knowledge, all amphibian hosts are new records for these nematode species, which are known to infect a wide range of amphibian and reptile species. Our findings suggest that nematode diversity is probably lower than previously assumed due to low host specificity. As morphological species identification is often challenging, our data facilitate molecular identification of adult and specifically larval nematodes found in amphibians of Sub-Saharan Africa.

Free-living amoeba (FLA) research in the Philippines is still in its infancy but has, by far, demonstrated the presence of potentially pathogenic species. Acanthamoeba may cause sight-threatening and central nervous system infections to humans, yet its epidemiologic distribution from local environmental sources is yet to be defined. The present study aimed to provide a baseline epidemiologic distribution of Acanthamoeba spp. in freshwater systems in the Philippines and establish potential pathogenicity of isolates through thermo-tolerance assay. A total of 63 water samples were collected from 13 freshwater systems all over the Philippine archipelago. The low-volume (50 ml) water samples were processed and cultured on non-nutrient agar lawned with Escherichia coli and observed for amoebic growth using light microscopy. Amoebic culture demonstrated 14.28% (9/63) positivity while further molecular testing of culture-positive plates using Acanthamoeba-specific primers demonstrated 100% (9/9) confirmation of Acanthamoeba species. Genotyping of Acanthamoeba isolates revealed T1, T3, T4, T5, T7, T11, and T15 genotypes. Thermo-tolerance assay demonstrated that T5 and T7 genotypes were potentially pathogenic strains. The evidence of environmental distribution of Acanthamoeba spp. in the freshwater systems in the Philippines and thermo-tolerance profile of isolates are significant aspects of amoeba study in public health and calls for initiatives in the dissemination of relevant information and the expansion of knowledge, awareness, and policies on pathogenic waterborne amoeba to mitigate, prevent, detect, and report cases of human infections.

In the present article, we report on the identification of Vermamoeba (Hartmannella) vermiformis as the etiological agent of a tissue infection close to the eye of a female patient. Laboratory examination revealed no involvement of any pathogenic bacteria or fungi in the tissue infection. V. vermiformis was identified by cultivation and morphology of trophozoites and cysts as well as phylogenetic analysis of nuclear 18S rDNA. The lesion improved in the course of 4 weeks by application of zinc paste.

Many case reports emphasize the fact that Free-Living Amoebae (FLA) can relatively easily get in contact with humans or animals. The presence of several facultative parasitic FLA in habitats related to human activities supports their public health relevance. While some strains of Acanthamoeba, Naegleria fowleri , Balamuthia mandrillaris and several other FLA have been described as facultative human pathogens, it remains controversial whether Vermamoeba vermiformis strains may have a pathogenic potential, or whether this FLA is just an incidental contaminant in a range of human cases. However, several cases support its role as a human parasite, either as the only etiological agent, or in combination with other pathogens. Additionally, a wide range of FLA is known as vectors of microorganisms (endocytobionts), hereby emphasizing their environmental significance. Among those FLA serving as hosts for and vectors of (pathogenic) endocytobionts, there are also descriptions of V. vermiformis as a vehicle and a reservoir of those endocytobionts. The involvement in animal and human health, the role as vector of pathogenic microorganisms and the pathogenicity in cell cultures, led to the assumption that V. vermiformis should be considered relevant in terms of public health and environmental health.

Free-living amoebae (FLA) such as Acanthamoebae, Naegleriae and Vermamoebae have been identified from natural environments. Among the protozoa living in rivers and ponds, the FLA may also be parasites for humans and animals. These potentially pathogenic FLA are widely distributed depending on the environmental and climatic conditions. The most abundant, potentially parasitic FLA in rivers and ponds are the Acanthamoebae. Other highly pathogenic FLA, such as the "brain-eating amoebae" (Naegleria fowleri), require higher water temperatures and are therefore to be found only in warmer climates. As well as being parasites for humans and animals, FLA are known as reservoirs of and vectors for different microorganisms. In some cases, these intracellular microorganisms proliferate inside their host amoebae without being digested. Those microorganisms being able to survive the intracellular conditions in the amoebal trophozoites and cysts benefit from their intracelluar life because they are protected to a certain degree from any hostile environment. Furthermore they are dispersed by the FLA within their environment. These intracellular organisms are called "endocytobionts". Among those organisms resisting the lysis within their amoebic host, there are obligate and facultative pathogenic microorganisms affecting the health of humans or animals. Especially if FLA are present in biofilms in rivers and ponds, e.g. in the littoral zone, there are many possibilities for the FLA to get in contact with other microorganisms. The resulting relationship of "host and endocytobiont" may lead to the development of pathogenicity or to the enhancement of virulence of the endocytobionts. This passage through FLA together with survival of the phagocytosis mechanisms and even the subsequent proliferation may be an essential factor for the development of virulence factors. Thus the relationship may contribute to evolutionary processes. The contact of humans with parasitic FLA or FLA harbouring pathogenic endocytobionts may even increase in the future as a result of human activities in recreational waters due to global warming. At the same time, a higher abundance of the FLA is to be expected following global warming. In general, changes of the climate and the environment may increase the importance of many infectious diseases or pathogenic microorganisms affecting people, domestic animals and wildlife. FLA may contribute to this prospective development either as parasitic FLA or as vectors of pathogenic endocytobionts. Therefore they have to be considered in terms of "public health" and "environmental health".

Free-living amoebae (FLA) are protozoa ubiquitously found in nature. As some species or strains of these FLA are pathogenic for humans and animals, they represent objects of medical and parasitological research worldwide. Storage of valuable FLA strains in laboratories is often time- and energy-consuming and expensive. The shipment of such strains as frozen stocks is cumbersome and challenging in terms of cooling requirements as well as of transport regulations. To overcome these difficulties and challenges in maintenance and transport, we present a new method to generate lyophilised samples of non-cyst-forming FLA (Ripella (Vannella) spp.) and cyst-forming FLA (Acanthamoeba spp.) strains which guarantees a simple mechanism for long-term storage at ambient temperature, as well as easy handling and/or shipment. The survival rate of all FLA lyophilisates after short-term storage (2 months) was comparable to the survival rate of freeze cultures of the respective strains. Furthermore, the viability of Acanthamoeba spp. cysts after storage for 29 months was 20 to 40% following lyophilisation and rehydration, with strain variation.

There are numerous case reports indicating that naked Free-Living Amoebae (FLA) can relatively easily get to humans or animals. The presence of pathogenic amoebae in habitats related to human activities supports the public health relevance of FLA. Acanthamoebae, Naegleria fowleri, Balamuthia mandrillaris and several other FLA have proved to be facultative human pathogenic microorganisms. Additionally, a wide range of FLA is known as vectors of pathogenic microorganisms (endocytobionts). Within their biocoenosis, FLA and fungi (and other microorganisms) live sympatrically. It is known that fungi serve as food sources for the phagocytotic active (mycophagous) trophozoite stages of FLA. In contrast, amoebophagous fungi may use FLA as prey organisms. Endoparasitic and predaceous fungi prove that there are numerous different interactions between FLA and fungi. The man-made introduction of suitable fungi into a habitat (soil or water) with human pathogenic FLA may overcome any ecological effects or limits. While nematophagous fungi have already been brought into action against harmful nematodes, the usage of amoebophagous fungi against FLA has not been widely considered. Nevertheless, the results from in vitro studies are promising concerning the targeted use of amoebophagous fungi as biological control measures against FLA in limited natural areas, in soil and in aquatic habitats.

Free-living amoebae (FLA) can be found both in natural aquatic environments and in artificial, man-made aquatic environments. For a long time, (naked) FLA were considered to be harmless protozoa of soil and water. However, research since the 1960s has demonstrated that FLA can be pathogenic to humans and animals with nearly 100% morbidity from some strains. As etiological agents of the so-called Acanthamoebiasis, the Acanthamoebae can trigger several specific diseases (or symptoms) in humans. The amoebiasis of the central nervous system is called granulomatous amebic encephalitis (GAE), when Acanthamoebae are the etiological agents, showing subacute to chronic progress. GAE differs clinically from the primary amoebic meningoencephalitis (PAM(E)), which is caused by Naegleria fowleri. The Acanthamoeba keratitis is not necessarily associated with an immune suppression, but rather with a trauma, exposure to contaminated water or, particularly, the improper handling of contact lenses, which promotes infection. The clinical picture of GAE by Balamuthia mandrillaris is characterized by headache and neck stiffness. The infection is chronic and the time between infection and appearance of neurological symptoms may range from one month to about two years. The prevalence of FLA in water networks is associated with biofilms, where the amoebae live within a biocoenosis sympatric with other microorganisms. These biofilms serve as feeding grounds for the FLA and provide protection to a certain degree while the FLA adhere to the surfaces. In such a biocoenosis there are multiple interactions between FLA and other microorganisms: In addition to their role as pathogens, FLA are known to serve as host of and vehicles for diverse intracellular organisms (bacteria, viruses, eucaryonts), some of them being natural human pathogens. They act as reservoir or vehicle for various microorganisms such as various Legionella sp., Listeria monocytogenes, Pseudomonas aeruginosa and some Mycobacterium sp. In the cyst-stage of the FLA these intracellular organisms (endocytobionts) are protected to a high degree against any adverse environment (FLA as “Trojan horse”). This host-endocytobiont relationship may further lead to health risks in terms of the development of pathogenicity/virulence and antibiotic resistance (FLA as “Trainings ground”).

Objective: FLA are predatory heterotrophic microorganisms, feeding as trophozoites on bacteria, cyanobacteria, fungi and algae while adhering to surfaces through phagocytosis. It is known that FLA produce and expel vesicles as part of the digestive process. Bacteria are packaged in multilamellar bodies and are released into the environment if not digested. In 2008, it was shown how easily the so-called Pandoraviruses can get in close contact with humans, while proliferating in Acanthamoeba . Materials and Methods: In our study, the search for these packages and multilamellar bodies in Acanthamoebae was conducted by electron microscopy with special emphasis on Pandoravirus inopinatum virions being involved in these processes. The multilamellar bodies were detected within the trophozoites of the amoeba host strain and as expelled vesicles within their environment. Neither digested, partially digested or viable Pandoravirus inopinatum virions nor developmental stages were found within these packages. A modified host range evaluation method was used to study the uptake and the proliferation of Pandoravirus inopinatum virions by Acanthamoeba trophozoites via light microscopy and to determine the host range. Results: In addition to the Acanthamoeba strain, which was found to harbor Pandoravirus inopinatum initially, we confirmed another 9 Acanthamoeba strains to be susceptible, among them members of genotypes T4 and T 11. Conclusion: The modified time series method, which we used, proved to be superior to the initial (more static) host range studies, in both axenic and xenic cultures.

An otherwise healthy 49-year-old female patient presented at the local hospital with severe keratitis in both inflamed eyes. She was a contact lens wearer and had no history of a corneal trauma. In our laboratory for medical parasitology Acanthamoebae were detected microscopically from the cornea scraping and from the fluid of the contact lens storage case after xenical culture and showed the typical cyst morphology of Acanthamoebae group II. The diagnosis of "Acanthamoeba keratitis" was established and successful therapy was provided. While the morphological microscopic method led to the correct diagnosis in this case, an in-house multiplex qPCR and a commercial qPCR showed false negative results regarding Acanthamoeba sp. The subsequent sequencing revealed the Acanthamoeba genotype T4. In the present case report, the inability to detect Acanthamoebae using qPCR only is presented. Therefore, we recommend the utilization of combined different assays for optimal diagnostic purposes.

Silver nanoparticles (AgNP) are used in a broad range of consumer products and industrial applications. During the regular product life cycle and disposal, AgNP are continuously released into the environment. Hence, the aim of this study was to investigate the potential ecotoxicological effects of AgNP exposure on amoebae. The Acanthamoeba castellanii ATCC 30234 strain and environmental isolate Acanthamoeba strain C5/2, which are both affiliated with genotype T4, were chosen as representatives of ecologically important soil protozoan organisms. The amoebae were exposed to citrate-stabilized AgNP (30 and 70 nm in size) for 24 h and 96 h at concentrations ranging from 600 µg L−1 to 20 mg L−1. A newly adopted cell culture based microscopic assay was applied to assess the adherence ability of the amoeba trophozoites. The general metabolic activity of Acanthamoeba was determined to be a second independent endpoint by means of intracellular reduction of the redox dye AlamarBlue®. The fate of AgNP within the amoebae and test solutions was visualized by light- and transmission electron microscopy (TEM). Both Acanthamoeba strains showed a significant dose-dependent decrease of adherence ability (p < 0.04) and metabolic activity (p < 0.01) after 96 h of AgNP exposure. The environmental Acanthamoeba strain C5/2 lost both its adherence ability and metabolic activity at lower AgNP concentrations than the type strain, indicating a higher sensitivity to ionic silver. This was confirmed by the application of AgNO3, provoking a higher effect level in strain C5/2. AgNP was visualized intracellularly by transmission electron microscopy within the cytoplasm of Acanthamoeba. This is the first report to show the ecotoxicological effects of short-term AgNP exposure on the soil protist Acanthamoeba, causing both changes in the adherence ability and metabolic activity of this amoeba. This combined approach may be a powerful tool in the future for predicting potential harmful ecotoxicological effects of AgNP exposure using soil protozoans.