First report of Eimeria myoxi in the garden dormouse (Eliomys quercinus Linnaeus, 1766) from Doñana Natural Area (Andalusia, SW Spain).

This work reports for the first time the presence and molecular characterization of Eimeria myoxi in the garden dormouse (Eliomys quercinus) from the Doñana Natural Area (Andalusia, SW Spain). Fresh faecal samples were collected from a total of 28 garden dormice, which were caught following current guidelines for the ethical use of animals in research, and processing by a standard flotation technique with saturated saline solution. Then, wet drops were examined microscopically, and the number of oocysts was semi-quantified. Eimeria oocysts were observed in 16 of the 28 (57.1%) faecal samples, showing most of them a very low number of oocysts (≤1 oocyst per microscopic field × 400). The unsporulated oocysts visualized in 16 faecal samples were subspherical and of length 19.2 ± 1.2 µm and width 17.4 ± 1.1 µm, being morphologically compatible with E. myoxi. This finding was supported by molecular analysis of the small subunit ribosomal RNA (SSU-rRNA) gene, identifying the same species in 22 of the 28 (78.6%) dormice, including 15 samples in which oocyst size was compatible with E. myoxi. Moreover, the subsequent analyses of the apicoplast open reading frame 470 (ORF470) and the mitochondrial cytochrome c oxidase subunit I (COI) genes confirmed the molecular identification of the isolates as E. myoxi. The phylogeny analyses were consistent with previous phylogenetic studies and support the existence of three lineages of rodent-infecting Eimeria species.

Gastrointestinal helminths in brown trout (Salmo trutta Linnaeus, 1758) captured in Galician rivers (NW Spain).

Specimens of Salmo trutta (n = 613) captured by local anglers in different rivers in Galicia (NW Spain) during the 2015 fishing season (15 March-15 August) were examined. In total 1479 adult helminths were recovered from the gastrointestinal tracts of 221 fish. Moreover, the microscopic observation of the sediments obtained, previous diphasic concentration, revealed the presence of helminth eggs in 485 trout specimens. The following species were identified by morphological and molecular analysis: Crepidostomum metoecus (8.97%) (Trematoda); Salmonema ephemeridarum (16.97%), Raphidascaris acus (9.46%) and Pseudocapillaria sp. (2.12%) (Nematoda); and Echinorhynchus truttae (8.48%) (Acanthocephala). The prevalence, mean intensity and mean abundance of each helminth species were determined in relation to size/age of the fish. The helminth infracommunity comprised a maximum of four species and the species richness was S = 5. The biological cycles of most of the helminth species recovered are dependent on benthic macroinvertebrate fauna, which, in turn, is influenced by the water quality. Therefore, any changes that take place in the aquatic ecosystem (due to anthropogenic activities or climate change) may be reflected in the helminth composition.

Molecular identification of Eimeria species in Spanish bats.

This is the first study reporting the detection and molecular characterization of Eimeria in bats in Spain, specifically in 12 of 32 chiropteran species described in the Iberian Peninsula. A total of 76 faecal samples were collected from different bat roosting sites across Spanish territory. The DNA was extracted from the samples and sequenced by targeting the Eimeria SSU-rRNA gene. Two Eimeria species were detected in 29 of the 76 faecal samples (38%), and the bat-specific Eimeria rioarribaensis and rodent-specific Eimeria jerfinica were detected in 4 (5%) and 25 (33%) of the samples, respectively. This is the first report of E. rioarribaensis in the bats Rhinolophus euryale, Myotis myotis and Nyctalus lasiopterus, extending the host and geographical ranges for this bat coccidian parasite. The identification of the rodent-specific parasite species E. jerfinica in bats indicates the occurrence of this species in Spain, although its presence has not previously been reported in wild rodents in this country. Considering that most of the Eimeria spp. reported in bats were described only on the basis of morphometric data, molecular studies are required to determined which Eimeria species occur in bats, to complete the identification of these species and to clarify the phylogeny.

A review of the current status of Cryptosporidium in fish.

Species of the genus Cryptosporidium (phylum Apicomplexa) infect the epithelium of the gastrointestinal tract of several vertebrate hosts, including humans and domestic and wild animals. In the past 20 years, several studies have focused on Cryptosporidium in fish. To date, a total of four piscine-host-specific species (Cryptosporidium molnari, Cryptosporidium huwi, Cryptosporidium bollandi and Cryptosporidium abrahamseni), nine piscine genotypes and more than 29 unnamed genotypes have been described in fish hosts. In addition, Cryptosporidium species and genotypes typical of other groups of vertebrates have also been identified. This review summarizes the history, biology, pathology and clinical manifestations, as well as the transmission, prevalence and molecular epidemiology of Cryptosporidium in wild, cultured and ornamental fish from both marine and freshwater environments. Finally, the potential role of piscine hosts as a reservoir of zoonotic Cryptosporidium species is also discussed.

Morphological and molecular characterization of a novel Myxobolus species from the gastrointestinal tract of brown trout (Salmo trutta) in Spain.

The genus Myxobolus Bütschli, 1882 is the largest group within the class Myxosporea and includes 905 nominal species, 18 of which have been found to infect fish belonging to the family Salmonidae. In the present study, microscopic analysis enabled detection of myxospores in 43 of 613 (7.0%) gastrointestinal tracts from brown trout (Salmo trutta) captured in several rivers in the northwest of the Iberian Peninsula. Measurement of the whole myxospores, polar capsules and other morphological characteristics, together with identification of the site of infection, has led us to propose a novel salmonid-myxobolid species, Myxobolus compostellanus n. sp. Molecular analysis of the small subunit ribosomal RNA (SSU-rRNA) gene yielded the same consensus sequence of 2039 bp in 14 fish specimens. A BLAST search indicated 97.6% similarity to Myxobolus neurobius. Phylogenetic analysis revealed that M. compostellanus n. sp. is clustered with other salmonid-infecting myxobolids. The present findings contribute to the existing knowledge about the genus Myxobolus, providing both morphological and molecular data on a novel species of Myxobolus found to infect the gastrointestinal tract of salmonids, M. compostellanus n. sp. in the brown trout (S. trutta).

Kinetic modeling of the synergistic thermal and spectral actions on the inactivation of Cryptosporidium parvum in water by sunlight.

Water contamination with the enteroprotozoan parasite Cryptosporidium is a current challenge worldwide. Solar water disinfection (SODIS) has been proved as a potential alternative for its inactivation, especially at household level in low-income environments. This work presents the first comprehensive kinetic model for the inactivation of Cryptosporidium parvum oocysts by sunlight that, based on the mechanism of the process, is able to describe not only the individual thermal and spectral actions but also their synergy. Model predictions are capable of estimating the required solar exposure to achieve the desired level of disinfection under variable solar spectral irradiance and environmental temperature conditions for different locations worldwide. The thermal contribution can be successfully described by a modified Arrhenius equation while photoinactivation is based on a series-event mechanistic model. The wavelength-dependent spectral effect is modeled by means of the estimation of the C. parvum extinction coefficients and the determination of the quantum yield of the inactivation process. Model predictions show a 3.7% error with respect to experimental results carried out under a wide range of temperature (30 to 45 °C) and UV irradiance (0 to 50 W·m-2). Furthermore, the model was validated in three scenarios in which the spectral distribution radiation was modified using different plastic materials common in SODIS devices, ensuring accurate forecasting of inactivation rates for real conditions.

Multilocus analysis reveals further genetic differences between Tritrichomonas foetus from cats and cattle.

Tritrichomonas foetus isolates from feline and bovine origin has been previously shown to carry a certain degree of genetic heterogeneity. Here, novel candidate molecular markers were developed by means of multilocus sequence typing of the gap2 gene (encoding for T. foetus glyceraldehyde-3-phosphate dehydrogenase), ITS region, the TR7/TR8 variable-length repeat and microsatellite genotyping. These markers were used to characterize T. foetus field isolates from bulls and domestic cats and to compare phylogenetically with the following ATCC isolates: T. foetus isolated from cattle and pig (syn. Tritrichomonas suis), Tritrichomonas mobilensis, Tetratrichomonas gallinarum and Pentatrichomonas hominis. Among them, TFMS10 and TFMS7 were found to be the most polymorphic markers. Moreover, an 809 bp fragment of the gap2 gene was successfully amplified from all the trichomonads included in this study and the sequence analysis revealed differences between T. foetus porcine and feline genotypes and T. mobilensis in comparison to the bovine T. foetus ATCC isolate. The TR7/TR8 repeat pattern was not reproducible, being only consistent the fragments of approximately 110 and 217 bp. Sequence analysis of the latter revealed the existence of 3 SNPs resulting in 98.6 % homology between bovine and feline isolates. A search for similar sequences was carried out to develop a Restriction Length Fragment Polymorphism analysis. A 503 bp region, named TF1, revealed the existence of two BbvI restriction enzyme sites that were able to generate different length fragments for T. foetus feline and bovine isolates. Finally, the neighbour-joining analyses showed that T. foetus porcine genotype clusters together with bovine genotype, whereas T. mobilensis and the feline genotype form a separate cluster.

Microbiological Evaluation of 5 L- and 20 L-Transparent Polypropylene Buckets for Solar Water Disinfection (SODIS).

BACKGROUND: Solar water disinfection (SODIS) is an appropriate technology for household treatment of drinking water in low-to-middle-income communities, as it is effective, low cost and easy to use. Nevertheless, uptake is low due partially to the burden of using small volume polyethylene terephthalate bottles (1.5-2 L). A major challenge is to develop a low-cost transparent container for disinfecting larger volumes of water. (2) Methods: This study examines the capability of transparent polypropylene (PP) buckets of 5 L- and 20 L- volume as SODIS containers using three waterborne pathogen indicators: Escherichia coli, MS2-phage and Cryptosporidium parvum. (3) Results: Similar inactivation kinetics were observed under natural sunlight for the inactivation of all three organisms in well water using 5 L- and 20 L-buckets compared to 1.5 L-polyethylene-terephthalate (PET) bottles. The PP materials were exposed to natural and accelerated solar ageing (ISO-16474). UV transmission of the 20 L-buckets remained stable and with physical integrity even after the longest ageing periods (9 months or 900 h of natural or artificial solar UV exposure, respectively). The 5 L-buckets were physically degraded and lost significant UV-transmission, due to the thinner wall compared to the 20 L-bucket. (4) Conclusion: This work demonstrates that the 20 L SODIS bucket technology produces excellent bacterial, viral and protozoan inactivation and is obtained using a simple transparent polypropylene bucket fabricated locally at very low cost ($2.90 USD per unit). The increased bucket volume of 20 L allows for a ten-fold increase in treatment batch volume and can thus more easily provide for the drinking water requirements of most households. The use of buckets in households across low to middle income countries is an already accepted practice.

First molecular data on Eimeria truttae from brown trout (Salmo trutta).

The genus Eimeria comprises obligate intracellular protozoan parasites belonging to the phylum Apicomplexa. Members of this genus cause enteric disease in a wide range of vertebrate hosts, including fish, reptiles, birds, and mammals. A total of 157 species of Eimeria that parasitize fish have been described; however, molecular information regarding these piscine parasites is scarce. In the present study, Eimeria oocysts were detected in 189 of 613 (30.8%) gastrointestinal tracts of brown trout (Salmo trutta) captured in several rivers in Galicia (NW Spain). Measurements of the sporulated oocysts, sporocysts, and other morphological characteristics enabled identification of the oocysts as Eimeria truttae. By molecular analysis of the small subunit ribosomal RNA (SSU-rRNA) gene, a single sequence of ~ 420 bp was obtained in 100 fish samples. After amplification of a ~ 1300-bp fragment of the same locus, two representative sequences that exhibited five nucleotide differences were obtained. Phylogenetic analysis grouped the samples within the piscine clade closest to Eimeria nemethi as they exhibited 96.7% similarity with this species. This study is the first to characterize E. truttae at the molecular level, thus helping to clarify the phylogenetic relationships between this and other Eimeria species isolated from fish and contributing further to the knowledge about this protozoan parasite.

First Report of Cryptosporidium molnari-Like Genotype and Cryptosporidium parvum Zoonotic Subtypes (IIaA15G2R1 and IIaA18G3R1) in Brown Trout ( Salmo trutta).

This study reports for the first time the molecular characterization of Cryptosporidium spp. in Salmo trutta. A total number of 613 brown trout was captured by local anglers in 44 Galician rivers within 10 river basins (NW Spain) during the 2015 fishing season (March-August) and classified into groups according to their size. The gastrointestinal tracts were dissected and differentiated in pyloric ceca and intestine, which were homogenized and concentrated in phosphate-buffered saline 0.04 M pH 7.2/diethyl ether (2:1). Cryptosporidium oocysts were observed by immunofluorescence microscopy in 103 of 613 specimens (16.8%), with a mean intensity of 326.7 oocysts/trout. The highest prevalence rate was detected in specimens <2 yr (23.1%). Considering the anatomical location, Cryptosporidium oocysts were observed in pyloric ceca (72 trout, 69.9%), intestine (15 trout, 14.6%), or in both locations (16 trout, 15.5%), showing statistically significant differences between the 2 locations ( P < 0.01). The prevalence rate in the pyloric ceca increased with the age/size of the fish (62.2% vs. 70.8% vs. 83.3% for trout <2, 2-3, and >3 yr, respectively). By contrast, the prevalence rate in the intestinal location decreased with the age/size of specimens (21.6% vs. 12.5% vs. 7.7% for trout <2, 2-3, and >3 yr, respectively), but statistically significant differences were not determined. The microscopic observation of clusters of 4-20 oocysts in the pyloric ceca from 5 specimens of 20-28-cm body length is remarkable. By polymerase chain reaction amplification and sequencing of fragments of small-subunit ribosomal DNA ( SSU-rDNA), GP60, hsp70, and actin loci, Cryptosporidium molnari-like genotype was identified in 1 trout and Cryptosporidium parvum (subtypes IIaA15G2R1 and IIaA18G3R1) in 47 fish, including those specimens in which oocyst clusters were observed. This finding may indicate a true infection by C. parvum, as the homogenization process would break the epithelial cells, releasing oocysts, free or in clusters. Cryptosporidium oocysts were detected in wild trout captured from 27 of 44 rivers sampled in Galicia (61.4%), belonging to 9 of the 10 river basins considered, confirming the presence of this protozoan parasite in Galician rivers and proving their wide dispersion in aquatic freshwater environments. The identification of the zoonotic species C. parvum in brown trout may indicate a risk to public health as trout may be a potential source of infection to humans. Thus, edible wild fish extend the range of foodstuffs involved in the transmission of cryptosporidiosis.

Use of ultrasound irradiation to inactivate Cryptosporidium parvum oocysts in effluents from municipal wastewater treatment plants.

Water reuse is currently considered an innovative way to addressing water shortage that can provide significant economic, social and environmental benefits, particularly -but not exclusively- in water deficient areas. The potential transmission of infectious diseases is the most common concern in relation to water reclamation. Cryptosporidium is an important genus of protozoan enteropathogens that infect a wide range of vertebrate hosts, including humans. The infective form (oocyst) is highly resistant to the environmental conditions and disinfection treatments. Consequently, Cryptosporidium is the most common etiological agent identified in waterborne outbreaks attributed to parasitic protozoa worldwide. The present study evaluates the efficacy of ultrasound disinfection, at three power levels (60, 80 and 100 W), pulsed at 50% or in continuous mode, for inactivating the waterborne protozoan parasite Cryptosporidium parvum in simulated and real effluents from municipal wastewater treatment plants (MWTPs). Overall interpretation of the results shows that the application of ultrasound irradiation at 80 W power in continuous mode for an exposure time of 10 min drastically reduced the viability of C. parvum. Thus, oocyst viabilities of 4.16 ±â€¯1.93%; 1.29 ±â€¯0.86%; 3.16 ±â€¯0.69%; and 3.15 ±â€¯0.87% were obtained in distilled water, simulated, real and filtered MWTP effluents, respectively (vs 98.57 ±â€¯0.01%, initial oocyst viability), as determined using inclusion/exclusion of the fluorogenic vital dye propidium iodide, an indicator of the integrity of the oocyst wall. Independently of the mode used (pulsed/continuous) and at 80 W power, higher level of oocyst inactivation was detected in MWTP effluents than in distilled water used as a control solution, may be due to the differences in the chemical composition of the samples. Comparison of the results obtained in both modes showed that use of the continuous mode yielded significantly lower oocyst viability. However, when the Dose parameter was considered (energy per volume unit), no statistically significant differences in oocyst viability were observed in relation to the type of mode used. The results demonstrate that ultrasound technology represents a promising alternative to the disinfection methods (ultraviolet irradiation and chlorine products) currently used in water reclamation as it drastically reduces the survival of Cryptosporidium oocysts, without changing the chemical composition of the water or producing toxic by-products.

Identification of a novel piscine Cryptosporidium genotype and Cryptosporidium parvum in cultured rainbow trout (Oncorhynchus mykiss).

This study reports for the first time the presence and molecular characterization of Cryptosporidium in farmed rainbow trout (Oncorhynchus mykiss Walbaum, 1792). A total of 360 fish, with no apparent clinical signs of disease, were collected and classified into groups according to their size. Cryptosporidium oocysts were detected by immunofluorescence microscopy in 33 specimens (9.2%), which were located in pyloric caeca samples (42.4%), intestinal scrapings (39.4%), or at both locations (18.2%). In the smallest (youngest) fish group, a higher percentage of positive samples were detected in the pyloric caeca relative to the intestinal location (58.8 vs. 17.6%; P = 0.01), including a cluster with more than 10 oocysts observed in the pyloric caeca of one specimen. PCR amplification and sequencing of fragments of SSU-rDNA and hsp70 genes identified a novel Cryptosporidium piscine genotype (genotype 9) in two specimens and Cryptosporidium parvum in seven fish, including the specimen in which the oocyst cluster was observed. Moreover, Cryptosporidium oocysts were detected in farm water samples (41.7 and 16.7% from influent and effluent, respectively). Although Giardia was not found in gastrointestinal samples, Giardia cysts were observed in 50.0 and 33.3% of the influent and effluent water samples, respectively. The results support the existence of natural infections by C. parvum in freshwater cultured fish, suggesting that the rainbow trout could shed infectious oocysts in aquatic environments and it may be a potential source of human infection when this edible fish is handled.

Detection of zoonotic and livestock-specific assemblages of Giardia duodenalis in free-living wild lizards / Detecção de genótipos de Giardia duodenalis zoonóticos e específicos de ruminantes domésticos em lagartos selvagens

Abstract Giardia duodenalis is a zoonotic parasite that infects the gut of a wide range of vertebrates, including numerous wildlife species. However, little is known about this protozoan parasite in reptiles. Fecal samples from 31 wild lizards were collected in Galicia (northwest Spain) and screened for the presence of Giardia by PCR amplification and sequencing of the ITS1-5.8S-ITS2 region in the ribosomal unit. This allowed detection of the parasite in 5 samples (16.1%), and enabled identification of G. duodenalis assemblage A2 in two samples of Iberian rock lizard (Iberolacerta monticola), G. duodenalis assemblage B in other two samples of I. monticola, and G. duodenalis assemblage E in one sample of Bocage's wall lizard (Podarcis bocagei). The results obtained after PCR amplification and sequencing of the SSU-rDNA gene confirmed the presence of G. duodenalis assemblage A in two samples of I. monticola. This is the first report of G. duodenalis in free-living lizards, although further studies are needed to distinguish between actual infection and mechanical dissemination of cysts. The detection of zoonotic and livestock-specific assemblages of G. duodenalis demonstrates the wide environmental contamination by this parasite, possibly due to human activities.

Resumo Giardia duodenalis é um parasito zoonótico que infecta o intestino delgado de uma ampla gama de vertebrados, sendo detectado em numerosas espécies selvagens. No entanto, pouco se conhece sobre a presença deste parasito protozoário em répteis. Para estudar a presença de Giardia, foram obtidas amostras fecais provenientes de 31 lagartos e coletadas em diferentes localizações de Galicia (Noroeste da Espanha). Mediante a aplicação da técnica de PCR e posterior sequenciamento da região ITS1-5.8S-ITS2 da unidade ribossômica, detectou-se Giardia em 5 amostras (16,1%), identificando-se o genótipo A2 de G. duodenalis em 2 amostras de lagartos da montanha (Iberolacerta monticola), G. duodenalis genótipo B em outras 2 amostras de I. monticola e G. duodenalis genótipo E em outra amostra de lagarto de Bocage (Podarcis bocagei). Os resultados obtidos, após amplificação e sequenciamento de um fragmento do gene SSU-rDNA, confirmam a presença de G. duodenalis genótipo A em 2 amostras de I. monticola. Esta é a primeira vez que se descreve G. duodenalis em lagartos selvagens, embora sejam necessários outros estudos complementares para confirmar se estes animais sofrem uma infecção real ou se apenas atuam como disseminadores mecânicos da contaminação ambiental. Além disso, a detecção de genótipos zoonóticos e específicos de ruminantes domésticos demonstra a contaminação do ambiente selvagem por G. duodenalis, possivelmente devido à atividade humana.

Detection of zoonotic and livestock-specific assemblages of Giardia duodenalis in free-living wild lizards.

Giardia duodenalis is a zoonotic parasite that infects the gut of a wide range of vertebrates, including numerous wildlife species. However, little is known about this protozoan parasite in reptiles. Fecal samples from 31 wild lizards were collected in Galicia (northwest Spain) and screened for the presence of Giardia by PCR amplification and sequencing of the ITS1-5.8S-ITS2 region in the ribosomal unit. This allowed detection of the parasite in 5 samples (16.1%), and enabled identification of G. duodenalis assemblage A2 in two samples of Iberian rock lizard (Iberolacerta monticola), G. duodenalis assemblage B in other two samples of I. monticola, and G. duodenalis assemblage E in one sample of Bocage's wall lizard (Podarcis bocagei). The results obtained after PCR amplification and sequencing of the SSU-rDNA gene confirmed the presence of G. duodenalis assemblage A in two samples of I. monticola. This is the first report of G. duodenalis in free-living lizards, although further studies are needed to distinguish between actual infection and mechanical dissemination of cysts. The detection of zoonotic and livestock-specific assemblages of G. duodenalis demonstrates the wide environmental contamination by this parasite, possibly due to human activities.

Evaluation of solar photocatalysis using TiO2 slurry in the inactivation of Cryptosporidium parvum oocysts in water.

Cryptosporidium is a genus of enteric protozoan parasites of medical and veterinary importance, whose oocysts have been reported to occur in different types of water worldwide, offering a great resistant to the water treatment processes. Heterogeneous solar photocatalysis using titanium dioxide (TiO2) slurry was evaluated on inactivation of Cryptosporidium parvum oocysts in water. Suspensions of TiO2 (0, 63, 100 and 200mg/L) in distilled water (DW) or simulated municipal wastewater treatment plant (MWTP) effluent spiked with C. parvum oocysts were exposed to simulated solar radiation. The use of TiO2 slurry at concentrations of 100 and 200mg/L in DW yielded a high level of oocyst inactivation after 5h of exposure (4.16±2.35% and 15.03±4.54%, respectively, vs 99.33±0.58%, initial value), representing a good improvement relative to the results obtained in the samples exposed without TiO2 (51.06±9.35%). However, in the assays carried out using simulated MWTP effluent, addition of the photocatalyst did not offer better results. Examination of the samples under bright field and epifluorescence microscopy revealed the existence of aggregates comprising TiO2 particles and parasitic forms, which size increased as the concentration of catalyst and the exposure time increased, while the intensity of fluorescence of the oocyst walls decreased. After photocatalytic disinfection process, the recovery of TiO2 slurry by sedimentation provided a substantial reduction in the parasitic load in treated water samples (57.81±1.10% and 82.10±2.64% for 200mg/L of TiO2 in DW and in simulated MWTP effluent, respectively). Although further studies are need to optimize TiO2 photocatalytic disinfection against Cryptosporidium, the results obtained in the present study show the effectiveness of solar photocatalysis using TiO2 slurry in the inactivation of C. parvum oocysts in distilled water.

Development of particulate drug formulation against C. parvum: Formulation, characterization and in vivo efficacy.

This research aims towards developing an alternative therapy against Cryptosporidium parvum using bioadhesive paromomycin and diloxanide furoate-loaded microspheres. Microspheres were prepared using chitosan and poly(vinyl alcohol) and two types of cyclodextrins (ß-CD and DM-ß-CD) for the potential use of treating cryptosporidiosis. This pathogen is associated with gastrointestinal illness in humans and animals. Microparticle formulations were characterized in terms of size, surface charge, drug release and morphology. In vivo bioadhesion properties of CHI/PVA microspheres were also evaluated in mice. Finally, the in vivo efficacy of CHI/PVA microspheres against C. parvum was tested in neonatal mouse model. In this work, microspheres prepared by spray-drying showed spherical shape, diameters between 6.67±0.11 and 18.78±0.07µm and positively surface charged. The bioadhesion studies demonstrated that MS remained attached at +16h (post-infection) to the intestinal cells as detected by fluorescence. This finding was crucial taking use of the fact that the parasite multiplication occurs between 16 and 20h post-infection. The efficacy of treatment was determined by calculating the number of oocysts recovered from the intestinal tract of mice after 7days of post-infection. Mice receiving orally administered microspheres with and without drug exhibited significantly lower parasite loads compared with the control mice. Ultrastructural observations by TEM bring to light the uptake of smallest particles by enterocytes associated with conspicuous changes in enterocytic cells. Completely recovery of cell morphology was detected after 24h of first inoculation with MS. CHI/PVA microspheres appear to be a safe and simple system to be used in an anticryptosporidial treatment. The distinctive features of neonatal mice requires further work to determine the suppressive effect of this particulate delivery system on C. parvum attachment in other animal models.

Occurrence of Giardia and Cryptosporidium in wild birds in Galicia (Northwest Spain).

Faecal samples were obtained from 433 wild birds being treated in wildlife recovery centres in Galicia (Northwest Spain), between February 2007 and September 2009. The birds belonged to 64 species representing 17 different orders. Giardia cysts and Cryptosporidium oocysts were detected by an immunofluorescence antibody test and identified at the molecular level by established PCR-sequencing methods. The overall prevalence of Giardia was 2·1% and that of Cryptosporidium, 8·3%. To our knowledge, this is the first description of Giardia sp. in Tyto alba and Caprimulgus europaeus; and of Cryptosporidium sp. in Apus apus, Athene noctua, C. europaeus, Falco tinnunculus, Morus bassanus, Parabuteo unicinctus and Strix aluco. Furthermore, the first PCR-sequence confirmed detection of Giardia duodenalis assemblage B in, Buteo buteo, Coturnix coturnix and Pica pica; G. duodenalis assemblage D in Garrulus glandarius; and G. duodenalis assemblage F in Anas platyrhynchos; Cryptosporidium parvum in Accipiter nisus, B. buteo, Milvus migrans, Pernis apivorus and P. pica; and Cryptosporidium meleagridis in Streptopelia turtur. The study findings demonstrate the wide spread of Giardia and Cryptosporidium between wild birds.

Giardia and Cryptosporidium in cetaceans on the European Atlantic coast.

The occurrence of Giardia and Cryptosporidium was investigated in cetacean specimens stranded on the northwestern coast of Spain (European Atlantic coast) by analysis of 65 samples of large intestine from eight species. The parasites were identified by direct immunofluorescence antibody test (IFAT) and by PCR amplification of the ß-giardin gene, the ITS1-5.8S-ITS2 region and the SSU-rDNA gene of Giardia and the SSU-rDNA gene of Cryptosporidium. Giardia and Cryptosporidium were detected in 7 (10.8 %) and 9 samples (13.8 %), respectively. In two samples, co-infection with both parasites was observed. Giardia duodenalis assemblages A, C, D and F, and Cryptosporidium parvum were identified. This is the first report of G. duodenalis in Balaenoptera acutorostrata, Kogia breviceps and Stenella coeruleoalba and also the first report of Cryptosporidium sp. in B. acutorostrata and of C. parvum in S. coeruleoalba and Tursiops truncatus. These results extend the known host range of these waterborne enteroparasites.

Benthic macroinvertebrate communities as aquatic bioindicators of contamination by Giardia and Cryptosporidium.

Benthic macroinvertebrates (community composed mostly by aquatic forms of insects, such as stonefly nymphs, dragonfly nymphs, water bugs or beetle larvae) are often used in biological monitoring programmes to evaluate the ecological status of rivers and thus to indicate the repercussions of anthropogenic activities. The aim of the present study was to evaluate the use of this indicator community to detect human enteroprotozoan parasites that are transmitted via water. In total, 32 samples of macroinvertebrates were collected, with the aid of surber nets of mesh size 500 µm, from nine rivers in Galicia (NW Spain), on different occasions between 2005 and 2009. The samples were homogenised (0.04 M phosphate buffered saline, pH 7.2), sieved (150 and 45 µm mesh), and concentrated (by a diphasic method). Aliquots of the sediments were then analysed by a direct immunofluorescence technique with monoclonal antibodies against Giardia and Cryptosporidium. Giardia cysts were detected in one (3.1%) of the samples and Cryptosporidium oocysts were detected in four (12.5%) of the samples. This work is the first study carried out to investigate the presence of Giardia and Cryptosporidium in this benthic community. The results demonstrate that benthic invertebrates could be used as bioindicators of contamination by these waterborne protozoans. Moreover, as this aquatic organisms act as intermittent accumulators and its monitoring enables chronological analysis of perturbations, in both the short- and mid-term, this may represent a suitable alternative or complementary method to the usual techniques of detecting human and animal enteropathogens in water samples.

In vitro evaluation of the suppressive effect of chitosan/poly(vinyl alcohol) microspheres on attachment of C. parvum to enterocytic cells.

We present a new strategy to suppress the attachment of Cryptosporidium parvum to the enterocytes cell surface by bioadhesive microspheres. An optimized microsphere system based on chitosan/poly(vinyl alcohol) was prepared by experimental design for the delivery of Diloxanide Furoate-cyclodextrin complex. Formulations were characterized in terms of size, surface charge, drug release, IR spectroscopy and morphology. Bioadhesion properties of chitosan/poly(vinyl alcohol) microspheres, evaluated in the human enterocytic HCT-8 model, were concentration and time dependent. In vitro efficacy of chitosan/poly(vinyl alcohol) microspheres against Cryptosporidium was tested in infected cultures and stages of parasite were assessed by immunofluorescence. The degree of adherence to cells and the inhibition of infectivity were directly related with the lowest level of cross-linking. The C. parvum attachment to cells surface was efficiently suppressed by a concentration of 100 µg/ml of microspheres. TEM observations showed no epithelial-cell damage when microspheres were co-incubated in infected cultures. These results were coincident with the lack of toxicity in cytocompatibility studies. Microspheres remained adhered after 72 h to the apical area of enterocytes. The results suggest that chitosan/poly(vinyl alcohol) with adequate size and appropriate surface characteristics suppress by impairment the attachment of sporozoites to enterocytes and may have a great potential in the oral chemotherapy of Cryptosporidium infections.