Novel Acanthamoeba 18S rRNA gene sequence type from an environmental isolate.

The free-living amoebae, Acanthamoeba, can act as opportunistic parasites on a wide range of vertebrates and are becoming a serious threat to human health due to the resistance of their cysts to harsh environmental conditions, disinfectants, some water treatment practices, and their ubiquitous distribution. Subgenus classification based on morphology is being replaced by a classification based on the sequences of the 18S rRNA gene with a total of 18 different genotypes (T1-T18). A new environmental strain of Acanthamoeba isolated from a waste water treatment plant is presented in this study as a candidate for the description of the novel genotype T19 after phylogenetic analysis.

Molecular characterization of Acanthamoeba isolated in water treatment plants and comparison with clinical isolates.

A total of 116 samples (44 clinical specimens and 72 environmental samples) have been analyzed for the presence of Acanthamoeba. The environmental samples (ESs) were collected from four drinking water treatment plants (DWTP, n=32), seven wastewater treatment plants (n=28), and six locations of influence (n=12) on four river basins from the central area of Spain (winter-spring 2008). Water samples were concentrated by using the IDEXX Filta-Max(®) system. Acanthamoeba was identified in 65 of the 72 ESs by culture isolation (90.3%) and 63 by real-time PCR (87.5%), resulting in all sampling points (100%) positive for Acanthamoeba when considering both techniques and all the time period analyzed. Nine of the 44 clinical specimens were positive for Acanthamoeba. Seventeen Acanthamoeba strains (eight from four DWTP and nine from clinical samples) were also established in axenic-PYG medium. Twenty-four of the ESs and the 17 Acanthamoeba sp. strains were genotyped as T4/1, T4/8, and T4/9. The eight strains isolated from the DWTP samples were inoculated in nude mouse to ascertain their potential pathogenicity in this model. Animals that were inoculated died or showed central nervous system symptoms 9 days post-inoculation. Examination of immunofluorescence-stained brain and lung tissue sections showed multiple organisms invading both tissues, and re-isolation of throphozoites was successful in these tissues of all infected animals. For the first time, potentially pathogenic Acanthamoeba T4 has been detected in 100% of different types of water samples including tap water and sewage effluents in the central area of Spain suggesting a potential health threat for humans especially for the contact lens wearers.

First cases of microsporidiosis in transplant recipients in Spain and review of the literature.

Microsporidia are currently considered emerging pathogens responsible for life-threatening infections in organ transplant recipients. Here, we describe the first cases of intestinal microsporidiosis by Enterocytozoon bieneusi genotype D in two non-HIV-infected renal transplant recipients from Spain. Previously reported cases of microsporidiosis in organ transplant recipients have also been reviewed, highlighting the necessity of considering organ transplant recipients a risk group for microsporidiosis. A systematic search for these parasites is recommended in cases of persistent diarrhea and in the differential diagnosis of other syndromes, such as chronic fever of unknown etiology.

High-level resistance of Nosema ceranae, a parasite of the honeybee, to temperature and desiccation.

Resistance of Nosema ceranae to different exposure conditions has been evaluated by using Sytox green and DAPI (4',6-diamidino-2-phenylindole) to test spore viability. High thermotolerance at 60 and 35 degrees C and resistance to desiccation were observed. However, a significant decrease in viability after freezing and a rapid degeneration of spores maintained at 4 degrees C were also detected.

Follow-up of antibody avidity in BALB/c mice infected with Toxocara canis.

In human Toxocara canis infection, an association has been shown between high IgG avidity in the chronic phase and low IgG avidity in recently acquired toxocarosis. The evolution of the antibody response in terms of avidity has been carried out through a T. canis infection in BALB/c mice. Infection with T. canis embryonated eggs (EE) was carried out with single doses (SD) of 6, 12, 50, 100, 200 or 1000 EE/mouse and with multiple doses (MD) of 200 and 1000 EE. Specific antibodies against T. canis (IgM+G, IgG, IgG1 and IgM) were detected by ELISA and Western Blot (WB) techniques in the presence and absence of urea. With the ELISA method, an increase in the avidity index (AI) of around 50% was detected from days 40-80 p.i. to the end of the study, with all the doses studied. The WB method showed the presence of high avidity antibodies bound to 100 kDa and 75 kDa T. canis proteins in all the cases when the IgM+G and the IgG1 antibodies were investigated. Antibodies of variable avidity were observed in those sera that recognized the group of low molecular weight proteins, between 37 kDa and 25 kDa.

Encephalitozoon microsporidia modulates p53-mediated apoptosis in infected cells.

Microsporidia are intracellular obligate parasites which have recently been found to be related to fungi. They have a unique extrusion apparatus that is able to inject the sporoplasm directly into the target cell without using receptors. Encephalitozoon microsporidia are a source of morbidity and mortality in humans. It has been suggested that microsporidia may modulate the host cell cycle and apoptosis. We report here that caspase-3 cleavage is inhibited at different times of Vero cell infection by Encephalitozoon microsporidia and that the phosphorylation and translocation of p53 to the nucleus, previous steps for the activation of this protein, do not occur after infection of Vero cells. Consequently, the transcriptional function of p53 is impaired during the infection cycle as demonstrated by luciferase reporter assays. Thus, to our knowledge, for the first time it is shown that an intracellular parasite may be able to multiply in the host cell without activating the p53 apoptotic pathway of that cell. However, changes in the expression of Bcl-2 or Bax levels were not observed.

Vectorial role of Acanthamoeba in Legionella propagation in water for human use.

Legionella spp. is the causative agent of Legionnaires' disease and is transmitted through aerosols emanating from man-made water systems. Legionella resistance to water treatments has been related to its association with environmental amoebae such as Acanthamoeba. Due to the high presence of this protozoon in Spain and the high rate of notification of Legionnaires' disease of this country, the aims of this work were to study the coexistence of these bacteria and protozoa in water as well as their interaction. The usefulness of Acanthamoeba co-culture for the isolation of environmental Legionella was also studied. For this purpose, 70 water samples were collected in 2011 from three Drinking Water Treatment Plants, three Wastewater Treatment Plants and five Natural Pools in Spain. Acanthamoeba was found by PCR in 87.1% (61/70) samples and, by culture in 85.7% (60/70) samples. Legionella was detected by PCR in 58.6% (41/70) of water samples, in 5.7% (4/70) by agar culture and 75.7% (53/70) by Acanthamoeba co-culture. From the 54 Acanthamoeba water isolates, Legionella was detected in 43 of them independently of Acanthamoeba's genotype (T3, T4 and T11). Legionella feeleii, Legionella birminghamiensis, Legionella gresilensis/berliardensis, Legionella fairfieldensis, Legionella drozanski and Legionella falloni were identified. In conclusion, our results showed that environmental Acanthamoeba is infected by Legionella to a high percentage, and due to its ubiquity, high resistance and its pathogenic potential per se, new methods for its elimination should be studied. Also, the high effectivity of Acanthamoeba co-culture for Legionella detection has been shown.

Cross-reactions of sera from Toxocara canis-infected mice with Toxascaris leonina and Ascaris suum antigens.

The ELISA method using larval excretory-secretory (E/S) products and homogenized Toxocara canis, Toxascaris leonina and Ascaris suum adult worm extract were used to determine possible cross-reactions in BALB/c and C57BL/10 mice, inoculated with embryonated eggs or adult worm extract of T. canis in single and multiple doses. When we used sera of mice infected with embryonated eggs of T. canis against different heterologous antigens, we observed no cross-reactions in BALB/c mice against A. suum E/S and adult worm extract antigens with a single dose. In multiple doses this was absent too against T. leonina adult worm extract in BALB/c mice, and in both strains against A. suum E/S and adult worm extract. In BALB/c mice inoculated with adult worm extract of T. canis we did not observe cross-reactions with A. suum E/S antigen with both inoculation doses. In the remainder of the experiments, we observed cross-reactions of different intensities.

Cross-reactions of sera from Toxascaris leonina and Ascaris suum infected mice with Toxocara canis, Toxascaris leonina and Ascaris suum antigens.

The ELISA method using larval ES products and homogenized Toxocara canis, Toxascaris leonina and Ascaris suum adult worms extract, was used to determine the possible cross-reactions in BALB/c and C57BL/10 mice inoculated with embryonated eggs or adult worms extract of T. leonina of T. leonina or A. suum in single and multiple doses. When we used sera of mice infected with embryonated eggs of T. leonina against different heterologous Ag, no cross-reactions against T. canis ES and A. suum ES Ag were observed using a single dose. Similarly, in multiple doses no response against T. canis ES Ag was observed. In mice inoculated with adult worms extract of T. leonina cross-reactions with T. canis ES and A. suum ES Ag did not occur. Sera from BALB/c mice infected with embryonated eggs of A. suum, was tested using ES Ag from both A. suum and T. canis and no reactions were observed. This fact confirmed the resistance of this murine strain to A. suum embryonated eggs. When we used sera of susceptible C57BL/10 mice infected against different heterologous Ag, we observed no cross-reactions against T. canis ES Ag. In the case of both BALB/c and C57BL/10 and C57BL/10 mice immunized with a single dose of A. suum adult crude extract no cross-reactions were seen against ES T. canis Ag and with sera from C57BL/10 mice against ES T. leonina. These facts confirmed the specificity of the ES T. canis Ag.

Persistence of immune response in human toxocariasis as measured by ELISA.

The antibody titer was followed in a group of patients, clinically diagnosed with toxocariasis, during a 5 year period. We observed that larvae can survive for at least 5 years in humans. Antigenic stimulation was enough to keep high levels of immunoglobulins over this period. Antibody levels decreased slowly and this pattern is similar to that shown by animal models.

Bilateral ocular toxocariasis demonstrated by aqueous humor enzyme-linked immunosorbent assay.

PURPOSE/METHODS: A 14-year-old girl with a history of contact with puppies and chronic bilateral panuveitis was examined. The fundus of the right eye showed an inferotemporal pars plana exudate. The fundus of the left eye showed an elevated posterior mass. RESULTS/CONCLUSION: Toxocara antibodies in the aqueous humor detected by enzyme-linked immunosorbent assay had a Goldmann-Witmer coefficient of 8.63 in the right eye and 8.94 in the left eye. Toxocariasis may be a cause of bilateral panuveitis.

Cross-reactivity between Anisakis simplex sensitization and visceral larva migrans by Toxocara canis.

The aim of this work was to study cross-reactivity in the diagnosis of two related ascaridosis. Nineteen patients diagnosed with recidivous acute urticaria (RAU) caused by Anisakis simplex and 26 patients diagnosed with visceral larva migrans (VLM) caused by Toxocara canis were studied employing commercial diagnostic kits and "in house" assay kits. Cross-reactivity observed was greater when using "in house" assay kits, suggesting that T. canis excretory-secretory antigens were not only recognized by antibodies from patients with RAU but with greater intensity compared to the A. simplex excretory-secretory antigens.

Microsporidiosis in travelers with diarrhea from the tropics.

BACKGROUND: Microsporidia are protozoa which mainly affect severely immunodepressed AIDS patients in developed countries as well as those in developing ones. Traveler's diarrhea affects approximately 40% of people traveling from industrialized countries to developing ones, and no pathogens are identified in many of those patients on their returning, suggesting that known enteropathogens escape detection or entirely new ones could be responsible. Very few reports of travel-related microsporidiosis have been described. METHODS: Between January, 1996 and January, 1998, a total of 40 European travelers from the tropics with a clinical picture of protected diarrhea (three or more loose stools per day lasting for more than 3 weeks) were evaluated. Weber's trichrome modified by Kokoskin stain for microsporidial spores were performed in stool samples of every patient. Microsporidial DNA extraction and PCR amplification were attempted in every stool sample where microsporidial spores were observed. RESULTS: Four cases of imported Enterocytozoon bieneusi were detected: one HIV-infected short term traveler, a pregnant long term traveler, and two immunocompetent short term travelers. Diarrhea was self-limited, and the spores cleared from the stools in all HIV-non infected travelers, but showed a chronic course in the HIV-infected one. CONCLUSIONS: Available data is too limited to affirm that residence or travel in tropical countries increases the risk for microsporidial infection, but the cases presented here suggest that E. bieneusi could be a cause of self-limited diarrhea in immunocompetent travelers returning from the tropics or could chronically affect immunocompromised HIV-infected travelers.

A year-long study of Cryptosporidium species and subtypes in recreational, drinking and wastewater from the central area of Spain.

A year-long longitudinal study was undertaken to evaluate the presence of Cryptosporidium spp. in drinking water treatment plants (DWTPs), wastewater treatment plants (WWTPs) and freshwater bathing beaches (FBBs) from the central area of Spain. Water samples were collected according to USEPA Method 1623, and concentrated by the IDEXX Filta-Max® system. Cryptosporidium species were detected based on PCR-restriction fragment length polymorphism and sequence analyses of the ssuRNA gene. C. hominis and/or C. parvum isolates were subtyped by DNA sequencing of the Gp60 gene. Among 150 samples, 23 (15.3%) were positive by IFAT and 40 (26.7%) by PCR. Cryptosporidium spp. was more frequent in WWTPs (26.2 and 50.8%) and FBBs (12.5 and 17.5%) by IFAT and PCR respectively. Effluent waters from DWTPs were negative for this parasite suggesting that they are suitable for public use. Tertiary treatment in the WWTPs demonstrated a high removal efficiency of Cryptosporidium in the samples evaluated. Cryptosporidium species identified included C. hominis, C. parvum, C. ubiquitum, C. andersoni and C. muris. Subtyping analysis revealed C. hominis IbA10G2 and IeA11G3T3 alleles, which is the first report of the latter in water samples. Cryptosporidium highest frequency was observed in winter and spring. Our data provide information about the occurrence and diversity of Cryptosporidium in water of human use from the central area of Spain.

A year long study of the presence of free living amoeba in Spain.

Free-living amoeba such as Acanthamoeba and Balamuthia mandrillaris can act as opportunistic parasites on a wide range of vertebrates and they are becoming a serious threat to human health due to the resistance of their cysts to harsh environmental conditions, disinfectants, some water treatment practices and their ubiquitous distribution. This work was carried out in order to study the presence of these free-living amoebae (FLA) and their possible seasonality in a continental-Mediterranean climate in different types of water. For this purpose, a total of 223 water samples were collected during one year from four drinking water treatment plants (DWTP), seven wastewater treatment plants (WWTP) and six locations of influence (LI) on four river basins from Spain. Water samples were concentrated using the IDEXX Filta-Max(®) system and analyzed by a triplex real time PCR that detects Acanthamoeba, B. mandrillaris and Naegleria fowleri. Agar plates were also seeded for Acanthamoeba culture. From the three FLA studied, N. fowleri was not detected in any sample while B. mandrillaris was found at the entrance of a DWTP; this being, to our knowledge, the first report of these protozoa in water worldwide. On the other hand, the presence of Acanthamoeba observed was higher, 94.6% of the studied points were positive by real time PCR and 85.2% by culture, resulting in 99.1% positive for Acanthamoeba with both methods. All genetically analyzed Acanthamoeba were genotype T4 but nine different T4/DF3 sequences were observed, three of them being described for the first time, assigning new codes. No seasonal distribution of Acanthamoeba was found. These facts should serve as a warning to contact lens wearers of the risk of a poor hygiene when handling their contact lenses. It should also serve as a signal to physicians to consider FLA as a possible causative agent of nervous system infections as well as Acanthamoeba keratitis due to their high environmental presence shown in this study.

Detection of zoonotic intestinal parasites in public parks of Spain. Potential epidemiological role of microsporidia.

Several studies have demonstrated that the soil of public parks presents an important source of infection which has a significant impact on public health. Children are the main group affected by accidentally ingestion of contaminated soil. This study was performed in order to identify the presence of zoonotic parasites in dog and cat faecal and soil samples from public parks of Madrid, Spain. Six hundred twenty-five and seventy-nine soil and faecal samples (presumably from dogs and cats) respectively were collected from 67 parks. Intestinal parasites were identified in 27 parks (40.3%), which were contamined with Giardia sp. (19.4%), microsporidia (19.4%), Toxocara spp. (16.4%), Cryptosporidium sp. (6%), Entamoeba histolytica (3%) and Ancylostomidae (3%). Combinations of two or more intestinal parasites were found in 11 parks, and it was common to find Giardia and microsporidia together in samples. Intestinal parasites were detected in 18% (112/625) of soil samples. The most frequent parasite species found in the examined soil samples were Toxocara spp. (16.4%), followed by Giardia sp. (4.5%) and Strongyloides sp. larvae (3%). The zoonotic parasites found in the 79 faecal samples were Giardia sp. (17.7%), Cryptosporidium sp. (9%), E. histolytica (2.5%), Trichuris vulpis (1.3%), Toxascaris leonina (1.3%) and microsporidia spores (28%). Microsporidia characterization by amplification of DNA confirmed 10 samples as positive, eight for E. bieneusi and two for E. hellem by PCR. The role of those parasites in the environment are discussed.

First detection and genotyping of human-associated microsporidia in pigeons from urban parks.

Microsporidia are ubiquitous opportunistic parasites in nature infecting all animal phyla, and the zoonotic potential of this parasitosis is under discussion. Fecal samples from 124 pigeons from seven parks of Murcia (Spain) were analyzed. Thirty-six of them (29.0%) showed structures compatible with microsporidia spores by staining methods. The DNA isolated from 26 fecal samples (20.9%) of microsporidia-positive pigeons was amplified with specific primers for the four most frequent human microsporidia. Twelve pigeons were positive for only Enterocytozoon bieneusi (9.7%), 5 for Encephalitozoon intestinalis (4%), and one for Encephalitozoon hellem (0.8%). Coinfections were detected in eight additional pigeons: E. bieneusi and E. hellem were detected in six animals (4.8%); E. bieneusi was associated with E. intestinalis in one case (0.8%); and E. hellem and E. intestinalis coexisted in one pigeon. No positive samples for Encephalitozoon cuniculi were detected. The internally transcribed spacer genotype could be completed for one E. hellem-positive pigeon; the result was identical to the genotype A1 previously characterized in an E. hellem Spanish strain of human origin. To our knowledge, this is the first time that human-related microsporidia have been identified in urban park pigeons. Moreover, we can conclude that there is no barrier to microsporidia transmission between park pigeons and humans for E. intestinalis and E. hellem. This study is of environmental and sanitary interest, because children and elderly people constitute the main visitors of parks and they are populations at risk for microsporidiosis. It should also contribute to the better design of appropriate prophylactic measures for populations at risk for opportunistic infections.

Evaluation of chemotherapy in experimental toxocarosis by determination of specific immune complexes.

Parasitism by the larval phase of Toxocara canis is a chronic process in which the larvae survive in the tissues, resulting in the constant stimulation of the immune system. As a result, the detection of specific antibodies may not reflect the active state of the parasite. We have studied the dynamics of the production of specific immune complexes by ELISA with the monoclonal antibody TC-1 in rabbits inoculated with single and multiple doses of T. canis eggs. We also compared this with the production of specific antibodies and their possible modification after treatment with mebendazole. The specific antibodies against excretory-secretory antigen were detected with peaks at 10 and 12 weeks depending on the dose and remained positive during the entire experiment (62 weeks). Treatment caused an increase in the level of detectable antibodies dropping to similar levels to the controls. Specific immune complexes were detected only in multiple doses, and were then positive during the entire experiment. From the beginning of treatment the values of immune complexes fell quickly, remaining at undetectable levels during the rest of the experiment. For this reason the detection of specific immune complexes is a valid technique for monitoring the efficiency of treatment.

Comparative study of assays detecting circulating immune complexes and specific antibodies in patients infected with Toxocara canis.

A sandwich ELISA method using previously described E/S antigen-specific monoclonal antibodies has been developed to detect circulating immune complexes in patients infected with Toxocara canis. This technique could be used for the study of the dynamics of the parasite-host relationship, as we believe the detection of immune complexes and/or soluble antigen to be an improvement over detection of antibodies only. In this parasitosis, antibodies may be present in residual levels for prolonged periods after active infection.

Serological evidence of toxocariasis in patients from Spain with a clinical suspicion of visceral larva migrans.

Sera from patients with clinical characteristics of toxocariasis were assayed using the ELISA method and larval excretory-secretory antigen. Four hundred and seven samples of Toxocara serology were received at the laboratory of Ciudad Sanitaria Juan Canalejo Hospital of Corunna, Spain, from 1984 to 1989. Of these, 30 were from adults, 332 from children and 45 from patients of unknown age, resulting in Toxocara seroprevalences of 23.3%, 32.8% and 17.7% respectively. The reasons for these serological differences in the rural and urban areas of Galicia, Spain are discussed.