In vitro interactions of Acanthamoeba castellanii Neff and Vibrio harveyi.

Free-living amoebae (FLA) are opportunistic protozoa widely distributed in the environment. They are frequently found in water and soil samples, but they have also been reported to be associated with bacterial human pathogens such as Legionella spp. Campylobacter spp or Vibrio cholerae among others. Including within Vibrio spp. V. harveyi (Johnson and Shunk, 1936) is a bioluminescent marine bacteria which has been found swimming freely in tropical marine waters, being part of the stomach and intestine microflora of marine animals, and as both a primary and opportunistic pathogen of marine animals. Our aim was to study the interactions between Vibrio harveyi and Acanthamoeba castellanii Neff. Firstly, in order to analyze changes in it cultivability, V. harveyi was coincubated with A. castellanii Neff axenic culture and with Acanthamoeba Conditioned Medium (ACM) at different temperatures in aerobic conditions. Interestingly, at 4 °C and 18-20 °C bacteria were still cultivable in marine agar, at 28 °C, in aerobic conditions, but there weren't significant differences comparing with the controls. We also noted an enhanced migration of Acanthamoeba toward V. harveyi on non-nutrient agar plates compared to controls with no bacteria.

Variation in Campylobacter jejuni culturability in presence of Acanthamoeba castellanii Neff.

Free-living amoebae (FLA) are protozoa that are widely distributed in the environment mainly in water and soil related habitats. These amoebae have also been reported to be associated with some bacterial pathogens for humans such as Campylobacter spp. The species C. jejuni is the causative agent of about 90% of human campylobacteriosis cases worldwide and this disease may even end up in severe autoimmune sequelae as Guillain-Barré syndrome (GBS). In this study, the interactions between the strain Acanthamoeba castellanii Neff and Campylobacter jejuni was investigated. Campylobacter jejuni was coincubated with Acanthamoeba castellanii Neff trophozoites at different temperatures, in order to evaluate the C. jejuni ability to grow in presence A. castellanii culture and Acanthamoeba Conditioned Medium (ACM). C. jejuni was coincubated with A. castellanii axenic culture at different temperatures in aerobic conditions. Our results revealed that bacteria were still cultivable (Blood Agar medium, at 37 °C, in microaerophilic atmosphere) after a 14 days C. jejuni - A. castellanii coculture, comparing with C. jejuni alone, which was only cultivable for 24 h.

Isolation and molecular characterization of a Naegleria strain from a recreational water fountain in Tenerife, Canary Islands, Spain.

Free-Living Amoebae (FLA) are widely distributed protozoa in the environment and have been isolated from many sources such as dust, soil and water. Among the pathogenic genera included in this group Acanthamoeba spp., Naegleria fowleri and Balamuthia mandrillaris have been reported to be causative agents of lethal encephalitis, disseminated infections and keratitis. Naegleria fowleri is a pathogenic FLA species which causes Primary Amoebic Meningoencephalitis (PAM). At present there are not many available data on the distribution of Naegleria species in Spain from environmental sources. Therefore, the aim of this study was to evaluate the presence of this genus in recreational water sources in the island of Tenerife, Canary Islands, Spain. In this study, ten samples collected from recreational water fountains were checked for the presence of Naegleria spp. using morphological and molecular identification tools. From the analysed samples, only one sample (seawater fountain) was positive for Naegleria spp. interestingly, not many reports of Naegleria spp. in seawater are available in the literature and thus awareness should be raised among the environmental and public health professionals.

Genotyping of clinical isolates of Acanthamoeba genus in Venezuela.

Free-living amoebae of Acanthamoeba genus are opportunistic pathogens distributed worldwide. Strains included in this genus are causative agents of a fatal encephalitis and a sight-threating keratitis in humans and other animals. In this study, 550 clinical samples which were collected between 1984 and 2014 from different patients with suspected infections due to Acanthamoeba were initially screened for the presence of this amoebic genus at the Laboratorio de Amibiasis-Escuela de Bioanálisis at the Universidad Central de Venezuela. Samples were cultured in 2% Non-Nutrient agar plates seeded with a layer of heat killed Escherichia coli. From the 550 clinical samples included in this study, 18 of them were positive for Acanthamoeba genus after culture identification. Moreover, positive samples were confirmed after amplification of the Diagnostic Fragment 3 (DF3) of the Acanthamoeba18S rDNA genus and sequencing was carried out in order to genotype the isolated strains of Acanthamoeba. Furthermore, the pathogenic potential of the strains was checked by performing thermotolerance and osmotolerance assays. Sequencing of the DF3 region resulted in the identification of genotype T4 in all the isolated strains. Moreover, most isolates were thermotolerant or both thermotolerant and osmotolerant and thus were classified as potentially pathogenic strains. To the best of our knowledge, this is the first report on the molecular characterization at the genotype level of Acanthamoeba strains in Venezuela.

High occurrence of Acanthamoeba genotype T4 in soil sources from Bolívar State, Venezuela.

Pathogenic strains of Acanthamoeba are causative agents of keratitis and encephalitis that often may end fatal in humans and other animals. In the present study, twenty-seven soil samples were collected in the Bolivar State in Venezuela and checked for the presence of Acanthamoeba. Samples were cultivated onto 2% non-nutrient agar plates seeded with a layer of heat killed E. coli. Amplification by PCR and sequencing of the DF3 region of the 18S rDNA of Acanthamoeba was carried out in order to confirm morphological identification of the amoebae. Furthermore, Acanthamoeba spp. was isolated from 51.8% of soil samples. Sequencing of the DF3 region of the 18S rDNA resulted in the identification of genotype T4 in all samples. To the best of our knowledge, this is the first report of genotype T4 in soil sources from Venezuela. Further studies should be carried out in this State and in the country in order to determine the current occurrence of Acanthamoeba in Venezuelan environments.

Isolation of thermotolerant Vermamoeba vermiformis strains from water sources in Lanzarote Island, Canary Islands, Spain.

In this study, twenty water samples were collected in the island of Lanzarote, Canary Islands, Spain in order to check for the presence of V. vermiformis strains in these samples. Water samples were cultured on 2% Non-Nutrient Agar (NNA) plates covered with a thin layer of heat killed E. coli and checked daily for the presence of Vermamoeba. After a week, V. vermiformis amoebae were observed in 2 of the 20 processed samples (10%) incubated at room temperature and 37°C. Molecular characterization was carried out by amplifying the 18S rDNA gene and DNA sequencing in order to confirm the identity of the isolated amoebic strains. To the best of our knowledge, this is the first report on the presence of FLA in environmental sources in Lanzarote Island and the first report of Vermamoeba vermiformis in water sources in this island. Furthermore, the two strains isolated in this study were collected in recreational areas with close contact with humans and thus awareness should be raised.

Isolation and Molecular Identification of Vermamoeba vermiformis Strains from Soil Sources in El Hierro Island, Canary Islands, Spain.

Free-living amoebae (FLA) are widely distributed protozoa in the environment and have been isolated from many sources such as dust, soil and water. Furthermore, some genera/species of FLA such as Naegleria fowleri, Balamuthia mandrillaris and Acanthamoeba spp. are also able to cause opportunistic infections in humans and other animals. More recently, FLA have been reported to be environmental carriers of pathogenic bacteria, fungi and viruses, and thus have gained further importance from the public health point of view. Among them, Acanthamoeba spp. and Vermamoeba vermiformis have been described in many occasions as the most common carriers of pathogens of high medical relevance such as Legionella pneumophila and Mycobacterium spp. In this study, 24 soil samples were collected from the island of El Hierro, Canary Islands, Spain, in order to check for the presence of V. vermiformis strains in these samples. Soil samples were cultured on 2 % non-nutrient agar plates covered with a thin layer of heat-killed E. coli and checked daily for the presence of Vermamoeba. After a week, V. vermiformis amoebae were observed in 5 of the 24 processed samples (20.8 %) incubated at room temperature and 37 °C. Molecular characterization was carried out by amplifying the 18S rDNA gene and DNA sequencing, confirming that the isolated strains belonged to Vermamoeba vermiformis species. The high percentage of V. vermiformis in the studied soil sources should raise awareness in the region since these amoebae are potential environmental carriers of pathogens of high medical relevance.

Acanthamoeba genotypes T2, T4, and T11 in soil sources from El Hierro island, Canary Islands, Spain.

The genus Acanthamoeba includes pathogenic strains which are causative agents of keratitis and encephalitis that often may end fatal in humans and other animals. In the present study, forty soil samples were collected in the island of El Hierro, Canary Islands, Spain, and checked for the presence of Acanthamoeba. Samples were cultivated onto 2 % non-nutrient agar plates seeded with a layer of heat killed Escherichia coli. Amplification by PCR and sequencing of the DF3 region of the 18S rDNA of Acanthamoeba was carried out in order to confirm morphological identification of the amoebae. Furthermore, Acanthamoeba spp. was isolated from 47.5 % of soil samples. Moreover, genotypes T2, T4, and T11 were identified in these samples. To the best of our knowledge, this is the first study to establish genotypes T2, T4, and T11 in soil sources from El Hierro island.

Isolation and molecular characterization of Acanthamoeba genotypes in recreational and domestic water sources from Jamaica, West Indies.

Free living amoebae (FLA) are amphizoic protozoa that are ubiquitous in nature. Infection with FLA may result in neurological, ocular and skin infections. Exposure to Acanthamoeba occurs frequently through water contact and knowledge of the presence of the organisms in water sources is important in understanding transmission dynamics. The distribution of Acanthamoeba was studied in recreational and domestic water samples collected from across Jamaica. Morphological assessment and polymerase chain reaction revealed Acanthamoeba spp. isolates in 50.6% (42/83) and 17.3% (14/81) of recreational and domestic water, respectively. Sequencing of the DF3 region of the 18S rDNA resulted in the identification of genotypes T3, T4, T5, T10 and T11 corresponding to Acanthamoeba spp: A. griffini, A. triangularis, A. lenticulata, A. culbertsoni and A. hatchetti. Moreover, T4 was the most frequently isolated genotype in both recreational and domestic water. Thermotolerance and osmotolerance assays indicated that most isolates were potentially pathogenic. This is the first report of T3 and T10 genotypes in the Caribbean and the first report of these Acanthamoeba spp. in Jamaican waters. The study shows that there is potential risk of infection to contact wearers who practise poor lens care. Further, Acanthamoeba should be considered as a cause of neurological infections in Jamaica.

Detection of Acanthamoeba on the ocular surface in a Spanish population using the Schirmer strip test: pathogenic potential, molecular classification and evaluation of the sensitivity to chlorhexidine and voriconazole of the isolated Acanthamoeba strains.

Pathogenic strains of Acanthamoeba are causative agents of a sight-threatening infection of the cornea known as Acanthamoeba keratitis, which is often associated with the misuse of contact lenses. However, there is still a question remaining to be answered, which is whether these micro-organisms are present on the ocular surface of healthy individuals. Therefore, the aim of this study was to determine the presence of Acanthamoeba on the ocular surface in healthy patients and also in those with other ocular surface infections. Sterile Schirmer test strips were used to collect samples from a group of patients who attended an ophthalmology consultation at the Hospital del Norte, Icod de los Vinos, Tenerife, Canary Islands. Most of the patients (46 individuals, 79.31  %) presented ocular surface pathologies such as blepharitis or conjunctivitis; the rest did not present any pathology. None of the patients included in the study wore contact lenses. The collected samples were cultured in 2  % non-nutrient agar plates and positive plates were then cultured in axenic conditions for further analyses. Molecular analysis classified all isolated strains as belonging to Acanthamoeba genotype tbl4, and osmotolerance and thermotolerance assays revealed that all strains were potentially pathogenic. Furthermore, all strains were assayed for sensitivity against voriconazole and chlorhexidine. Assays showed that both drugs were active against the tested strains. In conclusion, the Schirmer strip test is proposed as an effective tool for the detection of Acanthamoeba on the ocular surface.

A DEVH-box RNA Helicase from Leishmania braziliensis is Associated to mRNA Cytoplasmic Granules.

RNA helicases are ubiquitous enzymes that participate in almost all aspects of RNA processing, including RNA and RNA-protein complex remodelling. In trypanosomatids, which post-transcriptionally regulate gene expression, the formation of different kinds of ribonucleoprotein granules under stress conditions modulates the parasite's RNA metabolism. This paper describes the isolation of a putative DEVH-box RNA helicase produced by promastigotes of Leishmania braziliensis. Using a Cy3-labelled dT30 oligo, FISH showed the localization of this protein to mRNA granules under starvation stress conditions. The central region of the protein was shown to be responsible for this behaviour.

Statins and voriconazole induce programmed cell death in Acanthamoeba castellanii.

Members of the genus Acanthamoeba are facultative pathogens of humans, causing a sight-threatening keratitis and a life-threatening encephalitis. In order to treat those infections properly, it is necessary to target the treatment not only to the trophozoite but also to the cyst. Furthermore, it may be advantageous to avoid parasite killing by necrosis, which may induce local inflammation. We must also avoid toxicity of host tissue. Many drugs which target eukaryotes are known to induce programmed cell death (PCD), but this process is poorly characterized in Acanthamoeba. Here, we study the processes of programmed cell death in Acanthamoeba, induced by several drugs, such as statins and voriconazole. We tested atorvastatin, fluvastatin, simvastatin, and voriconazole at the 50% inhibitory concentrations (IC50s) and IC90s that we have previously established. In order to evaluate this phenomenon, we investigated the DNA fragmentation, one of the main characteristics of PCD, with quantitative and qualitative techniques. Also, the changes related to phosphatidylserine exposure on the external cell membrane and cell permeability were studied. Finally, because caspases are key to PCD pathways, caspase activity was evaluated in Acanthamoeba. All the drugs assayed in this study induced PCD in Acanthamoeba. To the best of our knowledge, this is the first study where PCD induced by drugs is described quantitatively and qualitatively in Acanthamoeba.

Molecular characterization of Acanthamoeba strains isolated from domestic dogs in Tenerife, Canary Islands, Spain.

The present study describes two cases of Acanthamoeba infections (keratitis and ascites/peritonitis) in small breed domestic dogs in Tenerife, Canary Islands, Spain. In both cases, amoebic trophozoites were observed under the inverted microscope and isolated from the infected tissues and/or fluids, without detecting the presence of other viral, fungal or bacterial pathogens. Amoebae were isolated using 2 % non-nutrient agar plates and axenified for further biochemical and molecular analyses. Osmotolerance and thermotolerance assays revealed that both isolates were able to grow up to 37 °C and 1 M of mannitol and were thus considered as potentially pathogenic. Moreover, the strains were classified as highly cytotoxic as they cause more than 75 % of toxicity when incubated with two eukaryotic cell lines. In order to classify the strains at the molecular level, the diagnostic fragment 3 (DF3) region of the 18S rDNA of Acanthamoeba was amplified and sequenced, revealing that both isolates belonged to genotype T4. In both cases, owners of the animals did not allow any further studies or follow-up and therefore the current status of these animals is unknown. Furthermore, the isolation of these pathogenic amoebae should raise awareness with the veterinary community locally and worldwide.

Evaluation of two commercially available immunological kits for the diagnosis of Helicobacter spp. in bottlenose dolphins (Tursiops truncatus).

Helicobacter pylori is considered to be responsible for the most common gastric infections in humans worldwide. In animals, other Helicobacter species are linked to gastritis with and without the presence of ulcers in their respective hosts. Moreover, gastric ulcers have been reported for decades in wild and captive dolphins. Clinical signs include lack of appetite, anorexia, abdominal tenderness, depression, and occasional unresponsiveness. In this study, serum and stool of nine bottlenose dolphins from Loro Parque collection Tenerife, Spain were examined for the presence of Helicobacter spp. The aim of our study was to evaluate the use of two commercially available kits for the detection of H. pylori in humans: a stool antigen immunoassay (Letitest H. pylori CARD) and a Western blot assay (EUROLINE-WB H. pylori) that were adapted to identify specific Helicobacter spp. antibodies in the tested Loro Parque bottlenose dolphin collection. The utility of these diagnostic kits for their application in dolphins is demonstrated, and their use in the future for the diagnosis of Helicobacter spp. in both wild and captive dolphins is proposed in this study.

Isolation and genotyping of acanthamoeba strains from soil sources from Jamaica, West Indies.

Acanthamoeba spp. are opportunistic pathogens that are ubiquitous in nature. Many species of this genus are responsible for a fatal encephalitis and keratitis in humans and other animals. Seventy-two soil samples were collected from the parishes across Jamaica and assessed for the presence of Acanthamoeba spp. Cultivation was carried out on non-nutrient agar plates seeded with heat killed Escherichia coli. PCR and sequencing of the DF3 region were carried out in order to genotype the isolated strains of Acanthamoeba. Thermotolerance and osmotolerance assays were utilized to investigate the pathogenic potential of the Acanthamoeba isolates. Acanthamoeba spp. was isolated from 63.9% of soil samples. Sequencing of the DF3 region of the 18S rDNA resulted in the identification of genotypes T4, T5, and T11. T4 genotype was most frequently isolated. Most isolates were thermotolerant or both thermotolerant and osmotolerant, indicating that they may present the potential to cause disease in humans and other animals.

A multisystemic Acanthamoeba infection in a dog in Tenerife, Canary Islands, Spain.

A 22-month-old male Spanish water dog was hospitalized after its physical examination revealed fever and movement difficulty. After 24h, the dog was found to have a high fever (39.5 °C) and was treated empirically with doxycycline/ciprofloxacin. At 48 h, after submission the fever rose to 41 °C and the animal presented with a stiff neck and dehydration. Peripheral blood and cerebrospinal fluid (CSF) were sampled and trophozoites with an Acanthamoeba-like morphology were observed in the CSF. PCR specific for Acanthamoeba, Naegleria fowleri and Balamuthia mandrillaris were performed and the CSF sample found positive for Acanthamoeba. Lungs, kidney, liver and spleen samples were collected post mortem. All collected organ samples were positive for Acanthamoeba by PCR, thus confirming a multisystemic infection. Water samples taken at a suspected site of infection yielded an almost identical PCR fragment to those of the clinical samples, indicating that this was probably where the infection originated. This is the first report of a fatal case of Acanthamoeba disseminated infection in a dog in Spain.

Isolation and molecular characterization of Acanthamoeba and Balamuthia mandrillaris from combination shower units in Costa Rica.

Free living amoebae (FLA) are ubiquitous protozoa, which may behave as parasites under certain conditions. Four genera are recognized as causal agents of infections in humans and animals: Naegleria, Sappinia, Acanthamoeba and Balamuthia. This work determines the presence of FLA in combination shower units and employs molecular biology for the characterization of isolates. The morphological analysis and partial sequencing of the 18S rDNA gene revealed the presence of Acanthamoeba genotype T4 in 30% of the units sampled. In addition to Acanthamoeba cysts, trophozoites with morphological characteristics similar to Balamuthia were identified. PCR assay using the mitochondrial 16S rRNA gene as a target confirmed the identification of the amoeba as Balamuthia mandrillaris. Up to date, this is the first report of the isolation of B. mandrillaris in Central America and the fifth report worldwide.

Development of an indirect immunofluorescence technique for the evaluation of generated antibody titers against Erysipelothrix rhusiopathiae in captive bottlenose dolphins (Tursiops truncatus).

Erysipelothrix rhusiopathiae is the causative agent of erysipelas, a disease of many mammalian and avian species, mainly swine and turkeys. In cetaceans, erysipelas is considered to be the most common infection in juvenile individuals, which have not been vaccinated. Moreover, the disease manifest in both forms, the dermatologic and the acute septicemic forms, has been reported in various species of dolphins and whales. It is difficult to diagnose erysipelas by currently available approaches. Moreover, it is mainly based on culture methods and also PCR methods, which are currently being developed. At the present stage, prophylactic approaches are based on antibiotic therapy and vaccination mostly with porcine erysipelas vaccines. In the present study, an Indirect Immuno Fluorescence method for the detection of dolphin antibodies levels against E. rhusiopathiae was developed and applied in two different groups of captive bottlenose dolphins (Tursiops truncatus) from Loro Parque (Tenerife, Canary Islands, Spain) and L'Oceanogràfic de Valencia (Valencia, Spain) in order to check the tittering levels of antibodies after application of porcine erysipelas vaccines in the studied dolphins.

Presence of potentially pathogenic free-living amoebae strains from well water samples in Guinea-Bissau.

Free-living amoebae (FLA) include opportunistic pathogens such as Naegleria fowleri, Balamuthia mandrillaris, and the genera Sappinia and Acanthamoeba. In this study, a survey was conducted in order to evaluate the presence of potentially pathogenic amoebic strains in water samples collected from wells located in the western part of Guinea-Bissau. The samples were left to precipitate for 48 hours and then the sediments were seeded on non-nutrient agar plates containing Escherichia coli spread and cultures were checked daily for the presence of FLA. Identification of FLA strains was based on the morphological and polymerase chain reaction (PCR) using the 18S rDNA or 16S mitochondrial rDNA genes in the case of Naegleria and Balamuthia genera, respectively. In the case of positive samples of Acanthamoeba, strains were further classified at the genotype level by sequencing the diagnostic fragment 3 (DF3) region located in the 18S rDNA gene as previously described. Sappinia sp. was not isolated during the study and thus, no molecular analysis was performed for this genus. The obtained results revealed the presence of Acanthamoeba (genotypes T3 and T4), Naegleria fowleri, and Balamuthia mandrillaris. To the best of our knowledge, this is the first report demonstrating the presence of FLA in water bodies from Guinea-Bissau and the first report on the isolation of Balamuthia mandrillaris from environmental sources in Africa.

The isolation of Balamuthia mandrillaris from environmental sources from Peru.

Balamuthia mandrillaris is an opportunistic free-living amoeba that has been reported to cause skin lesions and the fatal Balamuthia amoebic encephalitis (BAE) in humans and other animals. Currently, around 200 human BAE cases have been reported worldwide, although this number is considered to be underestimated. The highest number of BAE cases has been reported in the American continent, mainly in the southwest of the USA. Peru seems to be another hotspot for BAE with around 55 human cases having been identified, usually involving cutaneous infection, especially lesions in the central face area. The isolation of Balamuthia from environmental sources has been reported on only three prior occasions, twice from Californian soils and once from dust in Iran and so it seems that this amoeba is relatively rarely encountered in samples from the environment. We investigated that possibility of finding the amoebae in soil samples from different regions where clinical cases have been reported in Peru. Twenty-one samples were cultured in non-nutrient agar plates and were checked for the presence of B. mandrillaris-like trophozoites and/or cysts. Those samples that were positive for these amoebae by microscopic criteria were then confirmed by PCR amplification and DNA sequencing of the mitochondrial 16S rDNA gene of B. mandrillaris. We have detected the presence of B. mandrillaris in four samples collected in the regions of Piura (3) and Lima (1) where infection cases have been previously reported. We hypothesize that B. mandrillaris is present in Peru in soil and dust which therefore constitutes a source of the infection for the BAE cases previously reported in this country. Further studies should be carried out in the area to confirm the generality of this finding.