Mouse neutrophils release extracellular traps in response to Naegleria fowleri.

Naegleria fowleri is a free-living amoeba, which is able to infect humans through the nasal mucosa causing a disease in the central nervous system known as primary amoebic meningoencephalitis (PAM). Polymorphonuclear cells (PMNs) play a critical role in the early phase of N fowleri infection. Recently, a new biological defence mechanism called neutrophil extracellular traps (NETs) has been attracting attention. These structures represent an important strategy to immobilize and kill invading microorganisms. In this work, we evaluate the capacity of N fowleri to induce the NETs release by PMNs cells in mice in vitro and in vivo. In vitro: Neutrophils from bone marrow were cocultured with N fowleri trophozoites. In vivo: we employed a mouse model of PAM. We evaluated DNA, histone and myeloperoxidase (MPO) and the formation of NETs by confocal microscopy. Our results showed N fowleri induce both NETs and MPO release by PMNs cells in mice after trophozoite exposure, which increased through time, in vitro and in vivo. These results demonstrate that NETs are somehow associated with the amoebas. We suggest PMNs release their traps trying to avoid N fowleri attachment at the apical side of the nasal epithelium.

Occurrence of free-living amoebae in streams of the Mexico Basin.

Free-living amoebae (FLA) are protozoa that are widely distributed in nature. They are important in the cycling of nutrients in aquatic food chains, but their distribution in natural aquatic environments is not well known. We conducted a survey to determine the presence and distribution of FLA and their relation to some physicochemical parameters in streams of the Mexico Basin in Central Mexico. Thirty-two sites from 18 streams were sampled. Samples were centrifuged and cultured onto NNA-media to isolate amoebae. Identifications were based on morphology. The pathogenicity of Acanthamoeba isolates was tested. Oxygen saturation, temperature, pH, specific conductance, water flow, dissolved reactive phosphorus, nitrites, nitrates, ammonia, dissolved inorganic nitrogen and coliforms were determined. One hundred-and-twenty FLA representing 18 genera were identified. The most frequent genera were Vannella, Rosculus and Acanthamoeba. The frequency of potentially pathogenic FLA was low and only 3 Acanthamoeba isolates were invasive in mice. The highest species richness of FLA was found in streams located into agriculture activity areas and those close to small villages that discharge wastewater into them. Water temperatures were always below 17°C. Oxygen saturation and pH were within the limits for the growth of most FLA. The presence of Acanthamoeba and Naegleria was low; nevertheless, they include potentially pathogenic species and can act as vectors and reservoirs for microbial pathogens and can produce human infections.

Intranasal coadministration of Cholera toxin with amoeba lysates modulates the secretion of IgA and IgG antibodies, production of cytokines and expression of pIgR in the nasal cavity of mice in the model of Naegleria fowleri meningoencephalitis.

The nasal mucosa is the first contact with antigens to induce IgA response. The role of this site has rarely been studied. We have shown than intranasal administration with Naegleria fowleri lysates plus Cholera toxin (CT) increased the protection (survival up to 100%) against N. fowleri infection in mice and apparently antibodies IgA and IgG together with polymorphonuclear (PMN) cells avoid the attachment of N. fowleri to apical side of the nasal epithelium. We also observed that nasal immunization resulted in the induction of antigen-specific IgG subclasses (IgG1 and IgG2a) in nasal washes at days 3 and 9 after the challenge and IgA and IgG in the nasal cavity, compared to healthy and infected mice. We found that immunization with both treatments, N. fowleri lysates plus CT or CT alone, increased the expression of the genes for alpha chain, its receptor (pIgR), and it also increased the expression of the corresponding proteins evidenced by the ∼65 and ∼74kDa bands, respectively. Since the production of pIgR, IgA and IgG antibodies, is up-regulated by some factors, we analyzed the expression of genes for IL-10, IL-6, IFN-γ, TNF-α and IL-1ß by using RT-PCR of nasal passages. Immunization resulted in an increased expression of IL-10, IL-6, and IFN-γ cytokines. We also aimed to examine the possible influences of immunization and challenge on the production of inflammatory cytokines (TNF-α and IL-1ß). We observed that the stimulus of immunization inhibits the production of TNF-α compared to the infected group where the infection without immunization causes an increase in it. Thus, it is possible that the coexistence of selected cytokines produced by our immunization model may provide a highly effective immunological environment for the production of IgA, IgG and pIgR as well as a strong activation of the PMN in mucosal effector tissue such as nasal passages.

Naegleria fowleri glycoconjugates with residues of α-D-mannose are involved in adherence of trophozoites to mouse nasal mucosa.

We analyzed the possible role of glycoconjugates containing α-D-mannose and α-D-glucose residues in adherence of trophozoites to mouse nasal epithelium. Trophozoites incubated with 20 µg of one of three different lectins which preferentially recognized these residues were inoculated intranasally in Balb/c mice. Mouse survival was 40% with Pisum sativum and Canavalia ensiformis and 20% with Galanthus nivalis amebic pretreatment, compared with 0% survival for control animals administered trophozoites without pretreatment. Possibly some of the glycoproteins found in Naegleria fowleri represent an adherence factor. Differences in the saccharide sequences of the Naegleria species, even on the same glycoconjugate structure, could explain the different results corresponding to the distinct pretreatments (C. ensiformis, G. nivalis, and P. sativum). We found a higher expression of glycoconjugates recognized by P. sativum in Naegleria lovaniensis than N. fowleri, probably due to the higher number of oligosaccharides containing an α-1,6-linked fucose moiety expressed on the former species.

Saline-Tolerant Pathogenic Acanthamoeba spp. Isolated from a Geothermal Power Plant.

Few studies have been conducted in the cooling systems of power plants; they have focused on Naegleria fowleri, leaving a gap in the knowledge of other pathogenic free-living amoebae in this environment. The objective of this study was to determine the occurrence of saline-tolerant pathogenic Acanthamoeba in a geothermal power plant. The identification of isolated amoebae at genus level was carried out, observing their morphological characteristics; the determination of genotype and species of Acanthamoeba was performed via molecular biology (PCR). Water temperature ranged from 18 to 43 °C and conductivity from 4.0 × 104 to 8.7 × 104 µS/cm; this last value was greater than the seawater value. Only five amoeba genera were found. Acanthamoeba was in all the sampling sites, showing high saline tolerance. The high temperature, but mainly high conductivity, were the environmental conditions that determined the presence of pathogenic free-living amoebae in the hot water. All the strains of Acanthamoeba culbertsoni killed the mice, having a mortality of 40 to 100%. Acanthamoeba genotypes T10 and T5 were identified, T10 is rarely isolated from the environment, while T5 is more frequent. This is the first time that genotypes T5 and T10 have been reported in the environment in Mexico.

Acanthamoeba spp. in domestic tap water in houses of contact lens wearers in the metropolitan area of Mexico City.

A survey was carried out in the metropolitan area of Mexico City to determine the presence of Acanthamoeba in the tap water of houses of contact lens wearers. Water samples were taken from the mains water entry, bathroom sinks and storage containers (roof tanks, cisterns) of 27 houses; and from the solution contained in the contact lens cases. Samples were filtered and cultured onto NNE medium. The isolates were identified based on their morphological features and pathogenicity. Total and fecal coliforms, water temperature, pH, dissolved oxygen and residual free-chlorine were measured by standard methods. Forty five isolates of Acanthamoeba from 200 water samples were obtained. The highest number of amoebae was isolated from cisterns and roof tanks. Most Acanthamoeba isolates were non-pathogenic, however, their presence in tap water is a potential hazard since some species can cause Acanthamoeba keratitis and granulomatous amoebic encephalitis.

Identification of Immunogenic Antigens of Naegleria fowleri Adjuvanted by Cholera Toxin.

The intranasal administration of Naegleria fowleri lysates plus cholera toxin (CT) increases protection against N. fowleri meningoencephalitis in mice, suggesting that humoral immune response mediated by antibodies is crucial to induce protection against the infection. In the present study, we applied a protein analysis to detect and identify immunogenic antigens from N. fowleri, which might be responsible for such protection. A Western blot assay of N. fowleri polypeptides was performed using the serum and nasal washes from mice immunized with N. fowleri lysates, either alone or with CT after one, two, three, or four weekly immunizations and challenged with trophozoites of N. fowleri. Immunized mice with N. fowleri plus CT, after four doses, had the highest survival rate (100%). Nasal or sera IgA and IgG antibody response was progressively stronger as the number of immunizations was increased, and that response was mainly directed to 250, 100, 70, 50, 37, and 19 kDa polypeptide bands, especially in the third and fourth immunization. Peptides present in these immunogenic bands were matched by nano-LC-ESI-MSMS with different proteins, which could serve as candidates for a vaccine against N. fowleri infection.

Isolation and Identification of Naegleria Species in Irrigation Channels for Recreational Use in Mexicali Valley, Mexico.

Members of the genus Naegleria are free-living amoebae that are widely distributed in water and soil environments. Moreover, Naegleria fowleri is a pathogenic amoeba species that causes a fatal disease in the central nervous system known as primary amoebic meningoencephalitis (PAM) in humans. Since most reported infections due to N. fowleri are reported in recreational waters worldwide, this study was aimed to describe the presence of these amoebic genus in Mexicali Valley irrigation channels of recreational use. A total of nine water samples were collected and processed by triplicate, in nine different sites of the Valley. After filtering and culturing the samples, plates were examined, and the observed amoebae were morphologically identified at the genus level. In addition, the pathogenicity of these amoebic isolates was checked, and molecular characterization was performed by PCR/sequencing. The results revealed the presence of Naegleria spp. in all the channels sampled. Finally, molecular identification confirmed the presence of five different species of Naegleria: N. fowleri, N. australiensis, N. gruberi, N. clarki and N. pagei. The presence of these protists, particularly N. fowleri, should be considered as a potential human health risk in the region.

Amibas de vida libre y anemia aplástica grave en niños / Aplastic anemia and free-living amoeba in two children

Introducción. Las amibas de vida libre habitualmente viven como fagótrofas en el agua y suelo donde se alimentan de bacterias; también pueden producir infecciones del sistema nervioso central y otros tejidos en seres humanos y animales. La anemia aplástica es una entidad caracterizada por pancitopenia secundaria a disminución de la producción en médula ósea de todos los elementos formes de la sangre y ausencia de enfermedad primaria que infiltre, reemplace o anule la hematopoyesis activa. Casos clínicos. Se presentan 2 niños con historia de sangrados, pancitopenia y el diagnóstico de anemia aplástica grave por biopsia y aspirado de médula ósea, que finalmente fallecieron. Se muestran los hallazgos de amibas de vida libre en la médula. Conclusión. En estos casos no se encontró agente causal de la anemia aplástica, y se sugiere a las amibas como oportunistas del padecimiento.