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Although Cryptosporidium parvum , Giardia duodenalis and some Eimeria species are frequently involved in neonatal calf diarrhoea (NCD), detailed studies on their interactions are scarce. Therefore, a cross-sectional study including faecal samples from 404 diarrhoeic calves aged 0-30 days was performed. Cryptosporidium oocysts and G. duodenalis cysts were detected by immunofluorescence antibody test and positive samples were molecularly characterized. Eimeria oocysts were microscopically detected using the modified McMaster technique and morphometric species identification was performed. Cryptosporidium infections (53.7%) predominated over those by G. duodenalis (12.4%) and Eimeria spp. (6.9%). Three Cryptosporidium species were identified: C. parvum (98.0%), C. bovis (1.0%) and C. ryanae (1.0%). Eleven C. parvum zoonotic subtypes were detected, with IIaA15G2R1 as the most common; only G . duodenalis assemblage E was identified. Moreover, 10 Eimeria species were found, being Eimeria ellipsoidalis (96.4%), Eimeria bovis (85.7%), and Eimeria zuernii (71.4%) predominant. A significant positive association between G. duodenalis and Eimeria spp. was detected as well as a negative association between C. parvum and Eimeria spp. Our results showed that C. parvum wasprevalent in diarrhoeic calves under 21 days of life; Eimeria spp. and G. duodenalis were especially common in animals in their fourth week of life. Concurrent infections increased with age. Moreover, the study also revealed potential public health risks since a noticeable percentage of animals were infected with C. parvum zoonotic subtypes. Further studies are needed for determining the role of these parasites in co-infections with other enteropathogens and their implications in the pathogenicity of NCD., Competing Interests: No potential conflict of interest was reported by the author(s)., (© 2025 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.)

Purpose: The infection of brown trout (Salmo trutta) by the acanthocephalan parasite Echinorhynchus truttae is initiated by the ingestion of gammarid crustaceans harbouring the cystacanth form. Gammarus pulex has been reported as the common intermediate host of this parasite species. The absence of G. pulex in the rivers of Spain suggests that native gammarid species may play the role of intermediate host for E. truttae, which is the only acanthocephalan species reported in salmonids in this country. The aim of the present study was to investigate whether native gammarid species of Galician rivers can act as intermediate hosts of E. truttae., Methods: A total of 263 gammarid specimens were collected from rivers in Galicia and fixed in 70% ethanol. They were examined under stereomicroscope for the presence of acanthocephalan larval stages., Results: In one gammarid specimen identified as Echinogammarus lusitanicus, one cystacanth of Echinorhynchus truttae was observed., Conclusion: The presence of a cystacanth of E. truttae in the gammarid E. lusitanicus indicates that this species play a role as an intermediate host in the life-cycle of this parasite in Galicia, where G. pulex (the most frequently reported intermediate host of this parasite species) is lacking., Competing Interests: Declarations. Ethical Approval: Not applicable. Consent for Publication: Not applicable. Consent to Participate: Not applicable. Conflict of Interest: The authors declare that they have no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

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., Competing Interests: Declaration of Competing Interest The authors declare that there is no conflict of interest., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

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., Competing Interests: Declaration of Competing Interest None., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

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., Competing Interests: Declaration of Competing Interest None., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

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.

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).

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.

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.

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.

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 ± 1.93%; 1.29 ± 0.86%; 3.16 ± 0.69%; and 3.15 ± 0.87% were obtained in distilled water, simulated, real and filtered MWTP effluents, respectively (vs 98.57 ± 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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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.

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.

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., (Copyright © 2016 Elsevier B.V. All rights reserved.)

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.