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Eight Eimeria spp. (Apicomplexa: Eimeriidae) have been isolated from the ring-necked pheasant (Phasianus colchicus Linnaeus), native to the temperate zone of Asia and eastern regions of Europe. Enteric coccidiosis has become a major issue associated with the breeding of farmed pheasants for game bird release or meat production. In this study, 35 fecal samples were collected from two-to-three-month-old ring-necked pheasants from four pheasant-rearing farms in Ehime Prefecture, Japan. Microscopic examination using a saturated sugar solution technique detected numerous subspherical oocysts from the samples of one farm and ellipsoidal Eimeria phasiani Tyzzer, 1929 oocysts from the three other farms. The subspherical oocysts were artificially sporulated and measured 18.6 µm by 15.7 µm with a 1.18 shape index (n = 150). Each oocyst contained four 10.7 µm × 5.8 µm sporocysts (n = 30) and one coarse refractile polar granule; no micropyle or residua were detected. Each sporocysts contained two sporozoites with one large and one small refractile body and sparsely distributed residua. The complete, 1,443-bp cytochrome c oxidase subunit I gene (cox1) of this isolate exhibited low sequence identity with published Eimeria spp. sequences including E. phasiani that was previously recorded in the same area. Meanwhile, the oocyst morphology most closely resembled that of Eimeria tetartooimia Wacha, 1973, but with distinct refractile polar granules and sporocyst residua. The available GenBank cox1 sequence of E. tetartooimia exhibited a sequence identity of < 94.5% with the study isolate. Here, the coccidian isolate identified in this study represents a new Eimeria iyoensis n. sp. capable of infecting ring-necked pheasant., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

A major bottleneck in the progress of Cryptosporidium research is the lack of accessible cryopreservation of Cryptosporidium oocysts. Here, we present a protocol for the cryopreservation of Cryptosporidium isolates using enteroids. We describe the steps for the establishment of enteroid cultures and cryopreservation of C. parvum-infected HCT-8 cultures. We then detail procedures for the recovery and propagation of frozen parasites using enteroids. For complete details on the use and execution of this protocol, please refer to Deng et al. 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Coccidiosis represents a major driver in the economic performance of poultry operations, as coccidia control is expensive, and infections can result in increased feed conversion ratios, uneven growth rates, increased co-morbidities with pathogens such as Salmonella, and mortality within flocks. Shifts in broiler production to antibiotic-free strategies, increased attention on pre-harvest food safety, and growing incidence of anti-coccidial drug resistance has created a need for increased understanding of interventional efficacy and methods of coccidia control. Conventional methods to quantify coccidia oocysts in fecal samples involve manual microscopy processes that are time and labor intensive and subject to operator error, limiting their use as a diagnostic and monitoring tool in animal parasite control. To address the need for a high-throughput, robust, and reliable method to enumerate coccidia oocysts from poultry fecal samples, a novel diagnostic tool was developed. Utilizing the PIPER instrument and MagDrive technology, the diagnostic eliminates the requirement for extensive training and manual counting which currently limits the application of conventional microscopic methods of oocysts per gram (OPG) measurement. Automated microscopy to identify and count oocysts and report OPG simplifies analysis and removes potential sources of operator error. Morphometric analysis on identified oocysts allows for the oocyst counts to be separated into 3 size categories, which were shown to discriminate the 3 most common Eimeria species in commercial broilers, E. acervulina, E. tenella, and E. maxima. For 75% of the samples tested, the counts obtained by the PIPER and hemocytometer methods were within 2-fold of each other. Additionally, the PIPER method showed less variability than the hemocytometer counting method when OPG levels were below 100,000. By automated identification and counting of oocysts from 12 individual fecal samples in less than one hour, this tool could enable routine, noninvasive diagnostic monitoring of coccidia in poultry operations. This approach can generate large, uniform, and accurate data sets that create new opportunities for understanding the epidemiology and economics of coccidia infections and interventional efficacy., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

An Isospora species, Isospora amphiboluri, originally described by Canon in 1967 and later by McAllister et al. (1995), was isolated from a central netted dragon (Ctenophorus nuchalis) housed at a wildlife rehabilitation centre in Perth, Western Australia. Sporulated oocysts of Isospora amphiboluri (n = 30) are spherical, 24.2 (26.5-23.0) μm in length and 23.9 (22.4-25.9) μm in width, with a shape index of 1.01. The bilayered oocyst wall is smooth and light-yellow in color. Polar granule, oocyst residuum and micropyle are absent. The sporocysts are lemon-shaped, 15.7 (15.2-18.0) × 10.2 (8.9-11.2) μm, with a shape index (length/width) of 1.53. Stieda and substieda bodies are present, the Stieda body being small and hemidome-shaped and the substieda half-moon-shaped. Each sporocyst contains four vermiform sporozoites arranged head to tail. The sporozoites are 11.7 (9.9-16.2) × 3.0 (2.4-3.5) μm, with a shape index (length/width) of 3.87. A sporocyst residuum is present. Sporozoites contain a central nucleus with a finely distributed granular residuum. Comparison of oocyst measurements and their features with other valid Isospora species from hosts in the Agamid family confirmed that this Isospora species is Isospora amphiboluri. Molecular characterization of I. amphiboluri at the 18S rRNA and MTCOI loci showed the highest similarity with I. amphiboluri from the central bearded dragon, 99.8% and 99.7% respectively. This is the first report of I. amphiboluri from a central netted dragon in Australia., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Species in the genus Gorgoderina Looss, 1902 are parasites of the urinary bladder of amphibians and include around 50 species described globally. Molecular data on species of the genus are scarce, as is the information of their life-cycle patterns. During a survey on the genetic characterization of the frog trematodes in the tropical rain forest of Los Tuxtlas, in the Gulf of Mexico slope of Mexico, specimens of two morphotypes of Gorgoderina were sampled from the Rio Grande leopard frog, Rana berlandieri. One of them represented an undescribed species which is described herein as Gorgoderina rosamondae n. sp., whereas the other one was morphologically very similar to an apparently widely distributed North American species, G. attenuata, which has been previously reported in the same geographical area. Specimens of both morphotypes were sequenced for two nuclear and one mitochondrial genes. Phylogenetic trees corroborated the distinction of the new species, and data on the internal transcribed spacer 2 revealed genetic differences between G. attenuata sequenced from frogs in USA and specimens of Gorgoderina sp. from Los Tuxtlas, indicating the possibility that they also represent an undescribed species. COI sequences showed high genetic divergence values between the new species and Gorgoderina sp. from Los Tuxtlas (8.63-9.99%). Additionally, COI sequences of the larval forms (sporocyst, cercariae and metacercariae) sampled in the same locality from their first and second intermediate hosts (Pisidium sp. and Agriogomphus tumens, respectively) showed conspecificity, and the 3 host life-cycle of the new species was elucidated., (Copyright © 2021 Elsevier B.V. All rights reserved.)

We detected Eimeria oocysts from Japanese green pheasants (Phasianus versicolor) at a zoo in Osaka, Japan. The oocyst isolates were subspherical or ovoidal shaped and measured 17.2 (range 14.7-20.0) μm in length and 14.8 (13.3-16.7) μm in width with a length/width (L/W) ratio of 1.2 (1.0-1.4) and each had one polar granule. The oocysts lacked a residuum and micropyle. Sporocysts measured 9.8 (6.7-13.3) μm in length and 5.9 (4.7-7.3) μm in width, with a L/W ratio of 1.2 (1.1-1.4). Compared to previously published values, this strain shows morphological similarities with an isolate of E. teetartooimia from ring-necked pheasants from other countries. Phylogenetic analysis of the 18S rRNA and mitochondrial cytochrome c oxidase subunit I genes places the isolate in a clade related to chicken Eimeria spp., such as E. acervulina or E. brunetti. Although further analysis is needed, this information can be helpful for the diagnosis and determination of virulence of Eimeria spp. in pheasants., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Woodcreepers are passerines of the family Dendrocolaptidae, which have a high forest dependency. The current work aimed to redescribe Isospora striata McQuistion et al. 1997, from two new hosts in protected areas in Brazil, revealing new localities of parasitism, in addition to providing preliminary genotypic identifications via sequencing of the mitochondrial cytochrome c oxidase subunit 1 (COI) gene from both host species. Isospora striata has oocysts that are subspheroidal to ovoidal, 19.4 × 16.8 μm with smooth wall. Oocyst residuum is absent, but micropyle and polar granules are present. Sporocysts are ovoidal, 13.6 × 8.3 μm, with both Stieda and sub-Stieda bodies. Sporocyst residuum is present and sporozoites with refractile body, nucleus, and striations. The morphological study and the 100% similarity in sequencing of the COI gene between samples of different dendrocolaptid species confirmed the identification of a single species, supporting the identification of I. striata in the Brazilian Atlantic forest and consequently the wide distribution of this coccidian species in the Neotropical Region.

Introduction: Toxoplasma gondii is a protozoan parasite that has a widespread distribution among mammalians and birds. One of the reasons for the high prevalence may be due to ingesting oocyst disseminated by stray cats' feces. In Turkey, most of the citizens are closely associated with stray cats and they love to pet and feed them on the streets. In this study, we aimed to determine the prevalence of T. gondii DNA in feces of stray cats living in İzmir, Turkey in order to identify the transmission potential to humans and other animals., Methodology: Feces and blood samples of 465 stray cats were investigated for the presence of T. gondii oocysts by microscopy and for the presence of T. gondii DNA by two real time PCR methods. Furthermore, serum samples were analyzed for anti-T. gondii IgG antibodies using an ELISA., Results: Oocysts were detected in 0.43% of the stray cats by microscopy. T. gondii DNA was detected in 14.37% of the stray cats' feces samples. The seroprevalence rate was 37.84%. In the feces and/or blood PCR positive group, 35.89% of them were seropositive. Among the 176 seropositive cats, T. gondii DNA was detected in feces of 27 cats (15.34%)., Conclusions: This study first time showed the inter relation of T. gondii DNA in feces and blood samples and seropositivity. In sum, over 14% of the stray cats living outdoor may have an important role in transmission of toxoplasmosis to humans in İzmir as well as to other animals., Competing Interests: No Conflict of Interest is declared, (Copyright (c) 2021 Muhammet Karakavuk, Huseyin Can, Nebahat Selim, Berna Yesilsiraz, Evren Atli, Esra Atalay Sahar, Ferda Demir, Aytul Gul, Huseyin Gokhan Ozdemir, Nuray Alan, Mustafa Yalcin, Onur Ozkurt, Murat Aras, Tuncel Celik, Sengul Can, Aysu Degirmenci Doskaya, Adnan Yuksel Guruz, Mert Doskaya.)

Malaria parasites are fast replicating unicellular organisms and require substantial amounts of folate for DNA synthesis. Despite the central role of this critical co-factor for parasite survival, only little is known about intraparasitic folate trafficking in Plasmodium. Here, we report on the expression, subcellular localisation and function of the parasite's folate transporter 2 (FT2) during life cycle progression in the murine malaria parasite Plasmodium berghei. Using live fluorescence microscopy of genetically engineered parasites, we demonstrate that FT2 localises to the apicoplast. In invasive P. berghei stages, a fraction of FT2 is also observed at the apical end. Upon genetic disruption of FT2, blood and liver infection, gametocyte production and mosquito colonisation remain unaltered. But in the Anopheles vector, FT2-deficient parasites develop inflated oocysts with unusual pulp formation consisting of numerous single-membrane vesicles, which ultimately fuse to form large cavities. Ultrastructural analysis suggests that this defect reflects aberrant sporoblast formation caused by abnormal vesicular traffic. Complete sporogony in FT2-deficient oocysts is very rare, and mutant sporozoites fail to establish hepatocyte infection, resulting in a complete block of parasite transmission. Our findings reveal a previously unrecognised organellar folate transporter that exerts critical roles for pathogen maturation in the arthropod vector., (© 2020 The Authors. Cellular Microbiology published by John Wiley & Sons Ltd.)

Until now, two Sarcocystis species, S. cornixi and S. corvusi, were known to employ members of the family Corvidae as intermediate hosts. Between 2013 and 2019, having examined leg muscles of 23 common ravens in Lithuania, sarcocysts were detected in 18 birds (78.3%). Using light microscopy, transmission electron microscopy (TEM), and molecular analysis (three genetic loci, 18S rDNA, 28S rDNA, and ITS1), sarcocysts found in the common raven were described as a new species S. kutkienae. Under a light microscope, the observed sarcocysts were ribbon-shaped (1500-8147 × 53-79 μm) and had a wavy striated cyst wall that reached up to 1.5 μm. Lancet-shaped bradyzoites were 7.7 × 2.2 μm (6.1-9.0 × 1.2-3.0 μm) in size. Ultrastructurally, the sarcocyst wall was 1.5-1.8 μm in thickness and had conical-like protrusions with minute invaginations of a parasitophorous vacuolar membrane. The cyst wall was type 1e-like. Limited genetic variability was observed between the 18S rDNA and 28S rDNA sequences of S. kutkienae and other Sarcocystis spp. using birds as intermediate hosts. In contrast, S. kutkienae could be clearly identified by comparing sequences. At this locus, sequences of S. kutkienae shared the highest similarity (89.5-89.7%) with those of S. cornixi. Phylogenetic analysis showed that S. kutkienae was most closely related to Sarcocystis spp. that employs birds as intermediate and definitive hosts. The issue relating to which species might serve as definitive hosts of S. kutkienae in Lithuania is addressed.

Isospora parnaitatiaiensis Silva, Rodrigues, Lopes, Berto, Luz, Ferreira & Lopes, 2015 was identified from a new host, the plain antvireo Dysithamnus mentalis (Temminck), and also from the white-shouldered fire-eye Pyriglena leucoptera Vieillot, in its type-locality, the Itatiaia National Park in the southeastern Brazil, and a preliminary genotypic characterisation by sequencing the mitochondrial cytochrome c oxidase subunit 1 gene is provided. The oöcysts recovered from P. leucoptera and D. mentalis were polymorphic and have genotypic differences that were not considered sufficient for the description of new species, but only different genotypes and morphotypes of I. parnaitatiaiensis related to each host. These morphological and molecular variations were associated with a process of ongoing speciation and in adaptive development to their respective host species.

A new Eimeria species is described from a common bronzewing pigeon (Phaps chalcoptera) (Latham, 1790) in Western Australia. Sporulated oocysts of Eimeria chalcoptereae n. sp. (n = 30) are subspheroidal, 22-25 × 21-24 (23.5 × 22.6) μm; length/width (L/W) ratio 1.0-1.1 (1.04) μm. Wall bi-layered, 1.0-1.4 (1.2) μm thick, outer layer smooth, c.2/3 of total thickness. Micropyle barely discernible. Oocyst residuum is absent, but 2 to 3 small polar granules are present. Sporocysts (n = 30) ellipsoidal, 13-14 × 7-8 (13.5 × 7.2) μm; L/W ratio 1.8-2.0 (1.88). Stieda body present, flattened to half-moon-shaped, 0.5 × 2.0 μm; sub-Stieda present, rounded to trapezoidal, 1.5 × 2.5 μm; para-Stieda body absent; sporocyst residuum present, usually as an irregular body consisting of numerous small granules that appear to be membrane-bound. Sporozoites vermiform, with a robust refractile body and centrally located nucleus. Isolated Eimeria oocysts were analysed at the 18S and 28S ribosomal RNA and the mitochondrial cytochrome oxidase (COI) loci. Analyses revealed that Eimeria chalcoptereae n. sp. shared the highest number of molecular features with an Eimeria sp. previously identified from a domestic pigeon in Australia (KT305927-29), with similarities at these three loci of 98.53%, 97.32% and 94.93%, respectively. According to morphological and molecular analysis, the isolated coccidian parasite is a new species of Eimeria named Eimeria chalcoptereae n. sp. after its host, the common bronzewing pigeon (Phaps chalcoptera) (Columbiformes: Columbidae) (Latham, 1790).

A novel species of coccidia, resembling a member of the genus Eimeria, was found in bats, Scotophilus leucogaster, collected in southern Saudi Arabia has been described on the basis of unsporulated oocysts and DNA sequencing of the Internal Transcribed Spacer 1 (ITS1) and partial 18S rDNA regions. Unsporulated oocysts of this form are ovoidal to spheroidal and had a 2-layered wall, 1.5-2.0 (1.9 ± 0.2); the outer layer was light blue with striations, and thicker than the inner, darker layer. No micropyle was present. Unsporulated oocysts (N = 150) measured 27.2 × 22.1 (25-30 × 20-25), length width ratio, 1.2 (1.1-1.4). There was no evidence of an oocyst residuum and/or polar granule. This parasite was detected in 2/7 (29%) S. leucogaster collected from southern Saudi Arabia. Oocysts incubated at 25 °C in 2.5% K 2 Cr 2 O 7 did not sporulate after > 1 month. Unsporulated oocyst measurements were compared with other coccidian parasites of bats that discharge oocysts in their feces. Sequences of the ITS1 and the 18S rDNA regions obtained from the unsporulated oocysts grouped this coccidium from S. leucogaster with eimerian species from various rodent and squirrel species. It is critical that future investigators obtain fully sporulated oocysts of this coccidium for full description of the parasite recovered in our study so it can be correctly assigned to genus and given an accurate binomial.

From a longitudinal survey conducted on 30 Danish mink farms in 2016, 11.0% of faecal samples (456/4140) were positive for Cystoisospora laidlawi oocysts by microscopy, with 60% (189/315) of mink being positive at least once during the study period. Morphological analysis of sporulated oocysts identified Cystoisospora oocysts measuring 34.3 × 29.5 μm with an oocyst length/width (L/W) ratio of 1.2. The morphological features of the oocysts were identical to Isospora laidlawi previously morphological identified in farmed mink from Denmark and elsewhere. Phylogenetic analysis of 18S rDNA sequences (1221 bp) from three positive mink indicated that Cystoisospora from mink shared the highest genetic similarity to C. canis from a Canadian dog (99.6%). The phylogenetic analysis placed Cystoisospora from mink in a clade with other Cystoisospora isolates.

Coccidian parasites possess complex life cycles involving asexual proliferation followed by sexual development leading to the production of oocysts. Coccidian oocysts are persistent stages which are secreted by the feces and transmitted from host to host guaranteeing life cycle progression and disease transmission. The robust bilayered oocyst wall is formed from the contents of two organelles, the wall-forming bodies type I and II (WFBI, WFBII), located exclusively in the macrogametocyte. Eimeria nieschulzi has been used as a model parasite to study and follow gametocyte and oocyst development. In this study, the gametocyte and oocyst wall formation of E. nieschulzi was analyzed by electron microscopy and immuno-histology. A monoclonal antibody raised against the macrogametocytes of E. nieschulzi identified a tyrosine-rich glycoprotein (EnGAM82) located in WFBII. Correlative light and electron microscopy was used to examine the vesicle-specific localization and spatial distribution of GAM82-proteins during macrogametocyte maturation by this monoclonal antibody. In early and mid-stages, the GAM82-protein is ubiquitously distributed in WFBII. Few hours later, the protein is arranged in subvesicular structures. It was possible to show that the substructure of WFBII and the spatial distribution of GAM82-proteins probably represent pre-synthesized cross-linked materials prior to the inner oocyst wall formation. Dityrosine-cross-linked gametocyte proteins can also be confirmed and visualized by fluorescence microscopy (UV light, autofluorescence of WFBII).

Coccidian parasites of fish have received considerably less attention than their terrestrial counterparts, and within piscine hosts, most studies have focused on freshwater fish. The present study aimed to describe oocyst morphology, phylogenetic affinities, and the impacts of coccidian parasites infecting the internal organs of a commercially valuable marine fish, the blue whiting (Micromesistius poutassou), captured off the Portuguese coast. As part of the phylogenetic analysis, sequences from coccidians infecting the pout (Trisopterus luscus) and the Atlantic chub mackerel (Scomber colias) were included, and the oocyst morphology of the coccidians infecting the former was also reported. Results showed that the prevalence of coccidiosis in the blue whiting was very high (> 82%), occurring in all analyzed organs, despite being more abundant in the liver. A significant negative correlation was found between the abundance of the parasites in the liver and host condition index (p < 0.05), which indicates a negative effect on the fitness of this host. Phylogenetic analyses of the parasites found in all three species examined identified three different species of Goussia, closely related to Goussia clupearum. Adding to previous research, we propose the existence of a fourth group of Goussia, the clupearum type, able to infect multiple organs and phylogenetic related with G. clupearum.

Up to now, four coccidian species have been identified in Rallidae (Aves: Gruiformes): Eimeria mongolica, E. alakuli, E. paludosa and E. porphyrulae. Here, we described an Eimeria species, E. paludosa, from a common gallinule (Gallinula galeata) in Mexico. Oocysts were ovoid and wall pitted single-layered. A prominent micropyle was present, the oocyst residuum absent and the polar granule was present. On histological examination of tissues, endogenous stages (meronts, microgametocytes and macrogametocytes) were seen in the epithelial cells of the small intestine (upper and lower intestine). In addition to a new locality, this is the first description of E. paludosa from G. galeata and is the third description of a coccidian infecting Rallidae in the Americas.

The molecular divergence, morphology and pathology of a cryptic gregarine that is related to the bee parasite Apicystis bombi Lipa and Triggiani, 1996 is described. The 18S ribosomal DNA gene sequence of the new gregarine was equally dissimilar to that of A. bombi and the closest related genus Mattesia Naville, 1930, although phylogenetic analysis supported a closer relation to A. bombi. Pronounced divergence with A. bombi was found in the ITS1 sequence (69.6% similarity) and seven protein-coding genes (nucleotide 78.05% and protein 90.2% similarity). The new gregarine was isolated from a Bombus pascuorum Scopoli, 1763 female and caused heavy hypertrophism of the fat body tissue in its host. In addition, infected cells of the hypopharyngeal gland tissue, an important excretory organ of the host, were observed. Mature oocysts were navicular in shape and contained four sporozoites, similar to A. bombi oocysts. Given these characteristics, we proposed the name Apicystis cryptica sp. n. Detections so far indicated that distribution and host species occupation of Apicystis spp. overlap at least in Europe, and that historical detections could not discriminate between them. Specific molecular assays were developed that can be implemented in future pathogen screens that aim to discriminate Apicystis spp. in bees., (Copyright © 2020 The Authors. Published by Elsevier GmbH.. All rights reserved.)

In the current work, Isospora massardi Lopes, Berto, Luz, Galvão, Ferreira & Lopes, [10] is identified from white-necked thrushes Turdus albicollis Vieillot, 1818, rufous-bellied thrushes Turdus rufiventris Vieillot, 1818 and from a new host, the yellow-legged thrush Turdus flavipes (Vieillot, 1818) in a new locality, the Itatiaia National Park, in Southeastern Brazil, providing a preliminary genotypic characterization via sequencing of the mitochondrial cytochrome c oxidase subunit 1 (COI) and 18S small subunit ribosomal RNA genes. The oocysts and sporocysts of I. massardi of the current study are morphologically similar to the original description and are uniform in the proportionality of width on length, but exhibited different patterns of size associated with each host species. Furthermore, a genotypic difference of 3% was found in the COI sequences from T. flavipes and T. albicollis. Therefore, an ecological discussion is introduced aimed at associating these morphometric and genotypic differences with an ongoing speciation process., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The Javan ferret-badger Melogale orientalis (Carnivora: Mustelidae: Helictidinae) is a small carnivore endemic to Indonesia. In the family Mustelidae, 10 Eimeria, 12 Cystoisopora, one Isospora, and one Hammondia species are known, but no eimeriid coccidia has been yet described in the subfamily Helictinidae (ferret badgers). Coproscopic examination of Javan ferret-badgers imported into the Czech Republic revealed the presence of coccidian oocysts. Sporulated oocysts differ from other Eimeria known in the family Mustelidae by their small size (12.4-16.1 × 10.4-13.4 μm) and ovoidal shape. Morphological data and phylogenetic analyses of 18S rRNA and COI genes indicated a new species of Eimeria found in faecal samples of Javan ferret badgers. The species is described as E. melogale n. sp., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

A large-scale cross-sectional epidemiological study was conducted to evaluate prevalence, species diversity, and associated risk factors of Eimeria infections in 55 cattle farms across seven states of Colombia, including subtropical and tropical regions. In total, 1333 fecal samples from young animals (< 1 year of age) were examined at a single sampling date from August 2016 to December 2016. Flotation and McMaster techniques were conducted for parasitological investigation. Excreted Eimeria oocysts were allowed to sporulate in vitro and thereafter identified to species level based on morphological and morphometric characteristics. The overall Eimeria prevalence was 75.5% (1006/1333), with no difference observed between age categories. In total, 13 different Eimeria species were identified. The most prevalent species was E. bovis (33.5%), followed by E. auburnensis (12.5%) and E. zuernii (11.9%). Analysis of extrinsic associated risk factors revealed the floor type, feeding system, watering system, and herd size as significant (p < 0.05) risk factors for Eimeria spp. infections. Based on these data, it can be assumed that bovine coccidiosis infections occur ubiquitously in the country and might play an important role especially in its subclinical form by affecting production parameters in conventional cattle management systems.

We describe three new coccidian species of the genus Eimeria Schneider 1875 (Apicomplexa: Eimeriidae) and redescribe and report Eimeria zygodontomyis Lainson and Shaw, 1990 in the montane grass mouse, Akodon montensis Thomas, 1913 from the Serra dos Órgãos National Park in southeastern Brazil. Sporulated oocysts of Eimeria zygodontomyis are ellipsoidal to cylindrical with a 0.6 (0.5-0.8) μm thick very delicate bi-layered wall; length × width (n = 49) 18.3 × 12.5 (16-20 × 11-13) μm; length/width ratio of 1.4 (1.2-1.6); 1 polar granule occasionally present; micropyle, residuum both absent. Sporocysts are ellipsoidal; length × width 8.5 × 5.2 (8-11 × 5-6) μm; length/width ratio of 1.5 (1.3-1.7) μm; Stieda body is prominent; sub-Stieda body is absent; sporocyst residuum is compact. Sporulated oocysts of Eimeria montensis n. sp. are spheroidal to subspheroidal with a 1.2 (1.0-1.4) μm thick bi-layered wall; outer layer lightly pitted; length × width (n = 30) 16.3 × 12.5 (15-17 × 13-15) μm; length/width ratio of 1.3 (1.0-1.4); 1 polar granule present; micropyle, residuum both absent. Sporocysts are ellipsoidal; length × width 7.2 × 5.1 (6-8 × 4-6) μm; length/width ratio of 1.4 (1.2-1.6); Stieda body is present, sub-Stieda body is absent; sporocyst residuum consists of small, scattered granules. Sporulated oocysts of Eimeria uricanensis n. sp. are ovoidal to pyriform with a 1.4 ( 1.3-1.6) μm thick bi-layered wall; outer layer lightly pitted; length × width (n = 40) 26.6 × 18.6 (23-30 × 17-20) μm; length/width ratio of 1.4 (1.3-1.6); 1 polar granule present; micropyle, residuum both absent. Sporocysts are ellipsoidal, length × width 13.3 × 8.0 (10-16 × 7-9) μm; length/width ratio of 1.7 (1.5-1.9); Stieda body, sub-Stieda body both absent; sporocyst residuum consists of a cluster of granules, forming a spheroid mass. Sporulated oocysts of Eimeria parnasiensis n. sp. are subspheroidal to ellipsoidal with a 1.8 ( 1.3-2.4) μm thick bi-layered wall; outer layer lightly pitted; length × width (n = 54) 28.2 × 21.9 (26-32 × 19-28) μm; length/width ratio of 1.3 (1.2-1.4); 1 polar granule present; micropyle is absent; oocyst residuum is present and consists of a cluster of granules of varying thickness. Sporocysts are ovoidal, tapering towards the Stieda body; length × width 12.2 × 7.6 (10-13 × 6-9) μm; length/width ratio of 1.6 (1.4-1.9); Stieda body is present; sub-Stieda body is absent; sporocyst residuum is present and consists of an aggregate of thin granules.

Toxoplasma gondii tachyzoites and bradyzoites are studied extensively in the laboratory due to the ease with which they can be cultured. In contrast, oocysts and the sporozoites within them are more difficult to work with, in that cat infections are required for their generation and isolating sporozoites requires a laborious excystation procedure. More over some parasite species such as Hammondia hammondi are obligately heteroxenous and require passage through a cat for completion of the life cycle. There is no debate that there is great value in studying this important life cycle stage, and we present here a detailed description of the current protocols used in our laboratories to generate and isolate T. gondii and H. hammondi oocysts, and to excyst and purify the sporozoites within them for use in downstream experimental applications.

Coccidia (Chromista: Miozoa: Eimeriidae) of columbiform birds (Aves: Columbiformes) have been described since the end of the nineteenth century; however, some of these descriptions were poorly detailed or inconclusive. In this sense, the current work makes a detailed taxonomic revision reconsidering and organizing 18 Eimeria spp. and two Isospora spp. previously described or reported of Columbiformes. Along with this, a new species of Eimeria is morphologically and molecularly identified by the mitochondrial cytochrome c oxidase subunit 1 (COI) gene and by the 18S small subunit ribosomal RNA (18S) gene from the ruddy ground-dove Columbina talpacoti (Temminck, 1809) in the Médio Paraíba region of the State of Rio de Janeiro, southeastern Brazil. Eimeria columbinae n. sp. has subspheroidal oocysts, 14.7 × 13.2 μm, with smooth, bi-layered wall, ~ 1.1 μm and length/width ratio of 1.1. Micropyle and oocyst residuum are present, but polar granule is absent. Sporocysts are ellipsoidal to slightly asymmetrical, 9.0 × 5.1 μm, with both Stieda and sub-Stieda bodies. Sporocyst residuum present and sporozoites with refractile body and nucleus. This is the 19th description of an eimerian from Columbiformes in the World, and the second to have a molecular identification of the COI and 18S genes.

Cryptosporidiosis has been reported as an important cause of neonatal diarrhea and mortality in cattle, sheep, and other ruminants, but its impact on alpaca health has not been studied thoroughly. In this study, we have determined the prevalence and evaluated the role of cryptosporidiosis as a risk factor for diarrhea occurrence in newborn alpacas. During the calving season (January-March) of 2006, stool specimens (N = 1312) were collected from 24 herds of newborn alpacas in Puno and Cuzco, departments that account for the largest populations of alpacas in Peru. All the specimens were microscopically screened for Cryptosporidium spp. using the acid-fast technique. The association between Cryptosporidium detection and diarrhea was analyzed using χ 2 test and generalized lineal model. Cryptosporidium species were determined by PCR-RFLP analysis of the small subunit rRNA gene. Cryptosporidium oocysts were detected in 159 of 1312 (12.4%) newborn alpacas. Results of the analyses demonstrated that crypstosporidiosis was significantly associated with diarrhea (PR = 3.84; CI 95% 2.54-5.81; p < 0.0001). Only Cryptosporidium parvum was detected in the 153 Cryptosporidium-infected animals. Thus, there is an association of C. parvum infection with diarrhea in neonatal alpacas.

Background: Although more modern methods are available, quantitative PCR (qPCR) is reproducible, sensitive and specific with instruments and expertise readily available in many laboratories. As such, the use of qPCR in Cryptosporidium research is well established and still widely used by researchers globally. This method depends upon the generation of standards at different concentrations to generate standard curves subsequently used for the quantification of DNA., Methods: We assessed four types of DNA template used to generate standard curves in drug screening studies involving Cryptosporidium spp.: (i) serially diluted Cryptosporidium parvum oocysts (10 6 -1); (ii) diluted template DNA from pure oocysts (×10-×10 6 dilution of 10 6 oocyst DNA template); (iii) oocysts incubated in human ileocecal adenocarcinoma (HCT-8) cells (10 5 -1 and 5 × 10 4 -50); and (iv) diluted DNA template (5 × 10 4 ) from cell culture incubated parasites (×10-×1000)., Results: Serial dilutions of both cell culture and pure oocyst suspension DNA template yielded better linearity than cell culture derived standards, with dilutions of 10 6 oocysts exhibiting similar quantification cycle (Cq) values to those obtained from DNA template dilutions of 10 6 oocysts. In contrast, cell culture incubated oocysts demonstrated significantly higher DNA content than equivalent freely suspended oocysts and diluted DNA template from both cell culture derived and freely suspended oocysts across numerous concentrations., Conclusions: For many studies involving Cryptosporidium, only relative DNA content is required and as such, the superior linearity afforded by freely suspended oocysts and diluted DNA template (from either cell culture derived standards or freely suspended oocysts) will allow for more accurate relative quantification in each assay. Parasite division in the cell culture standards likely explains the higher DNA content found. These standards, therefore, have the potential to more accurately reflect DNA content in cell culture assays, and despite more modern methods available for absolute quantification, i.e. droplet digital PCR (ddPCR), the ubiquity of qPCR for the foreseeable future encourages further investigation into the reduced linearity observed in these standards such as varying oocyst seeding density, non-linear growth rates and assay efficiency.

Sporulated oocysts from the feces of infected cats with Toxoplasma gondii can cause detrimental disease in both humans and animals. To investigate the prevalence of feral cats that excrete T. gondii oocysts in the feces, we examined fecal samples of 563 feral cats over a 3-year period from 2009 to 2011. Oocysts of T. gondii excreted into the feces were found from 4 of 128 cats in 2009 (3.1%) and one of 228 (0.4%) in 2010 while none of the 207 cats in 2010 were found positive with oocysts in their feces, resulting in an overall prevalence rate of 0.89% (5/563) between 2009 and 2011. Among the 5 cats that tested positive with T. gondii oocysts, 4 of the cats were male and 1 was a female with an average body weight of 0.87 kg. Numerous tissue cysts of 60 µm in diameter with thin (&lt;0.5 µm) cyst walls were found in the brain of one of the 5 cats on necropsy 2 months after the identification of oocysts in the feces. A PCR amplification of the T. gondii-like oocysts in the feces of the positive cats using the primer pairs Tox-5/Tox-8 and Hham34F/Hham3R confirmed the presence of T. gondii oocysts in the feces. This study provides a good indication of the risk assessment of feral cats in the transmission of T. gondii to humans in Korea.

Isospora phainopepla n. sp. is described from the faeces of Phainopepla nitens (Swainson) collected in Joshua Tree National Park, California, USA. To our knowledge, there are no metazoan or protist parasites described yet from any species in the Ptiliogonatidae of the Americas. Sporulated oöcysts of the isosporan are ellipsoidal, 30-35 × 22-29 (32.9 × 25.4) μm, with a length/width (L/W) ratio 1.20-1.35 (1.30); one rice-shaped polar granule is present, but both oöcyst residuum and micropyle are absent. Sporocyts are elongate-ellipsoidal, 22-27 × 11-14 (23.9 × 12.0) μm, with L/W ratio 1.97-2.02 (2.00); prominent Stieda and sub-Stieda bodies are both present, but para-Stieda body is absent. Sporocyst residuum of many scattered granules is present and the sporozoites have prominent anterior and posterior refractile bodies. This parasite is clearly new to science and represents the first species of Isospora hosted by a New World bird species from this small family of silky flycatchers (Ptiliogonatidae). This article was registered in the Official Register of Zoological Nomenclature (ZooBank) as 7F664B0D-DF0E-4CAD-8CEA-45D79C8BBF92. This article was published as an Online First article on the online publication date shown on this page. The article should be cited by using the doi number. This is the Version of Record.

Isospora toxostomai n. sp. (Apicomplexa: Eimeriidae) is described based on material from the curved-billed thrasher Toxostoma curvirostre (Swainson) in the Central Highlands of Mexico. The new species possesses subspherical oöcysts, with a smooth, bi-layered wall. Sporulated oöcysts measure 22-25 × 21-24 (23.4 × 22.3) µm; length/width (L/W) ratio of 1.0-1.1 (1.1). Sporocysts are ellipsoidal, 15-17 × 10-11 (15.8 × 10.5); L/W ratio of 1.3-1.6 (1.5). Micropyle and oöcyst residuum are both absent, and a polar granule present (many fibrils). Mean dimensions of both sporulated oöcysts and sporocysts of I. toxostomai n. sp. appear to be considerably larger than those of Isospora mimusi Coelho, Berto, Neves, Oliveira Flausino & Lopes, 2011 from the tropical mockingbird Mimus gilvus (Vieilot) in Brazil. This is the second species of Isospora Schneider, 1881 infecting a host of the Mimidae in the Americas.

Kinesin-8 proteins are microtubule motors that are often involved in regulation of mitotic spindle length and chromosome alignment. They move towards the plus ends of spindle microtubules and regulate the dynamics of these ends due, at least in some species, to their microtubule depolymerization activity. Plasmodium spp. exhibit an atypical endomitotic cell division in which chromosome condensation and spindle dynamics in the different proliferative stages are not well understood. Genome-wide shared orthology analysis of Plasmodium spp. revealed the presence of two kinesin-8 motor proteins, kinesin-8X and kinesin-8B. Here we studied the biochemical properties of kinesin-8X and its role in parasite proliferation. In vitro, kinesin-8X has motility and depolymerization activities like other kinesin-8 motors. To understand the role of Plasmodium kinesin-8X in cell division, we used fluorescence-tagging and live cell imaging to define its location, and gene targeting to analyse its function, during all proliferative stages of the rodent malaria parasite P. berghei life cycle. The results revealed a spatio-temporal involvement of kinesin-8X in spindle dynamics and an association with both mitotic and meiotic spindles and the putative microtubule organising centre (MTOC). Deletion of the kinesin-8X gene revealed a defect in oocyst development, confirmed by ultrastructural studies, suggesting that this protein is required for oocyst development and sporogony. Transcriptome analysis of Δkinesin-8X gametocytes revealed modulated expression of genes involved mainly in microtubule-based processes, chromosome organisation and the regulation of gene expression, supporting a role for kinesin-8X in cell division. Kinesin-8X is thus required for parasite proliferation within the mosquito and for transmission to the vertebrate host., Competing Interests: The authors have declared that no competing interests exist.

Using a combination of morphological and molecular data, we describe a new apicomplexan parasite, Isospora svecica sp. n., from the white-spotted bluethroat, Luscinia svecica cyanecula, from the Czech Republic. Oocysts were found in its intestinal tract. Sporulation was exogenous and took 1-3 days. The oocysts were slightly ellipsoidal, of average size 26.17 × 20.33 μm, with a smooth bilayered wall. Micropyle, oocyst residuum, and polar granules were absent. Sporocysts were bottle-shaped, of an average size of 18.82 × 8.82 μm, with a thin, colourless wall. A conspicuous knob-like Stieda body was present. Substieda body was barely visible. Sporocyst residuum was present in the form of granules of various sizes. Sporozoites were banana-shaped and contained large anterior and small posterior refractile bodies. Partial DNA sequences of three genes were obtained from oocysts of Isospora svecica sp. n., being most closely related to other isosporans described from passerines. Little is known about the parasites of the avian family Muscicapidae, including coccidia, a highly prevalent parasitic protist group in all vertebrate classes. Only six species of the genus Isospora have so far been described in Muscicapidae, together with several "Isospora sp." that in fact most likely represent Isospora lacazei. The newly described Isospora svecica sp. n. differs morphologically from other coccidia reported from muscicapid birds, and represents the first coccidian species described from Luscinia svecica.

While nine nominal species of Eimeria Schneider, 1875 have been described from strigiform birds, molecular sequence data are not available for any of these species. In the present study, oöcysts of a coccidian were isolated by faecal flotation from the lower intestinal contents of an opportunistically collected, recently deceased great horned owl Bubo virginianus (Gmelin), sporulated in potassium dichromate, and subjected to morphological and molecular characterisation. Comparisons of morphological data with previous accounts of Eimeria spp. from owls were consistent with Eimeria megabubonis Upton, Campbell, Weigel & McKown, 1990. Novel molecular data for the 18S ribosomal RNA gene region and the mitochondrial cytochrome c oxidase subunit 1 gene are provided. The results of phylogenetic analysis based on concatenated sequence data from these regions are presented and implications for the evolutionary history of Eimeria are discussed.

Background: The human infectious reservoir for malaria consists of individuals capable of infecting mosquitoes. Oocyst prevalence and density are typical indicators of human infectivity to mosquitoes. However, identification of oocysts is challenging, particularly in areas of low malaria transmission intensity where few individuals may infect mosquitoes, and infected mosquitoes tend to have few oocysts. Here, features that differentiate oocysts from other oocyst-like in mosquito midguts are explained and illustrated. In addition, the establishment and maintenance of infrastructure to perform malaria transmission experiments is described. This work may support other initiatives to set up membrane feeding infrastructure and guide oocyst detection in low transmission settings., Methods: In 2014, an insectary was developed and equipped in Tororo district, Uganda. A colony of Anopheles gambiae s.s. mosquitoes (Kisumu strain) was initiated to support infectivity experiments from participants enrolled in a large cohort study. Venous blood drawn from participants who were naturally infected with malaria parasites was used for membrane feeding assays, using 60-80 mosquitoes per experiment. Approximately 9-10 days after feeding, mosquitoes were dissected, and midguts were stained in mercurochrome and examined by light microscopy for Plasmodium falciparum oocysts and similar structures. In supportive experiments, different staining procedures were compared using in vitro cultured parasites., Results: A stable colony of the Kisumu strain of An. gambiae s.s. was achieved, producing 5000-10,000 adult mosquitoes on a weekly basis. Challenges due to temperature fluctuations, mosquito pathogens and pests were successfully overcome. Oocysts were characterized by: presence of malaria pigment, clearly defined edge, round shape within the mosquito midgut or on the peripheral tissue and always attached to the epithelium. The main distinguishing feature between artifacts and mature oocysts was the presence of defined pigment within the oocysts., Conclusions: Oocysts may be mistaken for other structures in mosquito midguts. Distinguishing real oocysts from oocyst-like structures may be challenging for inexperienced microscopists due to overlapping features. The characteristics and guidelines outlined here support identification of oocysts and reliable detection at low oocyst densities. Practical advice on sustaining a healthy mosquito colony for feeding experiments is provided. Following the reported optimization, the established infrastructure in Tororo allows assessments of infectivity of naturally infected parasite carriers.

Isospora sporophilae Carvalho-Filho, Meireles, Ribeiro & Lopes, 2005 was morphologically and molecularly identified from the double-collared seedeater Sporophila frontalis (Verreaux), which is categorised as 'vulnerable' by the International Union for Conservation of Nature and Natural Resources (IUCN), and from the uniform finch Haplospiza unicolor Cabanis in conserved and anthropomorphic/fragmented areas of Atlantic Forest in the southeastern Brazil. The oöcysts recovered from S. frontalis and H. unicolor had small morphological and genotypic differences that were not considered sufficient for the description of new species, but only different genotypes of I. sporophilae related to each host. This coccidian species was originally described from double-collared seedeaters Sporophila caerulescens (Vieillot) in a center screening of wild animals; therefore, this new report emphasises a potential occurrence of anthropomorphic dispersion of coccidia through illegal trade, seizures and reintroductions in the wild.

Human cryptosporidiosis is caused primarily by two species of apicomplexan parasites, Cryptosporidium parvum and C. hominis. Although infection of cell monolayers with sporozoites does not support the complete parasite life cycle, the in vitro system is used to study the asexual phase of multiplication, which consists of two generations of merogony. To better understand host-parasite interaction and to gain insight into gene regulatory processes driving the complex life cycle of Cryptosporidium parasites, we analyzed the transcriptome of C. parvum in oocysts, sporozoites and infected cell monolayers 2-48 h post-infection. Analysis of RNA-Seq data from replicate oocyst, sporozoite and intracellular samples revealed significant differences between transcriptomes expressed outside and inside the host cell. Compared to the transcriptome found in the host cell, the oocyst transcriptome is less diverse. Biological processes significantly over-represented intracellularly relate to biosynthetic processes. Genes significantly overexpressed in oocysts show evidence of specialized functions not found in other Apicomplexa. A more comprehensive view of gene regulation during the Cryptosporidium life cycle will require the analysis of later time points during the infection, particularly of the poorly studied sexual phase of the life cycle.

In November 2017, oöcysts of the coccidian Eimeria macyi Wheat, 1975 were isolated from the faeces of a single eastern red bat Lasiurus borealis Müller in Lowndes County, Mississippi, USA. Sporulated oöcysts, morphologically consistent with previous accounts of E. macyi in other chiropterans, were spherical to sub-spherical in shape with a highly mamillated outer wall that appears bi-layered. Oöcysts allowed to sporulate in 2.5% potassium dichromate at ambient temperature (c.23°C) for 7 days were 17-25 × 15-20 (20.7 × 17.9) µm. Micropyle and oöcyst residuum were absent with one to two polar granules scattered among sporocysts. The four ovoid sporocysts were 7-12 × 6-8 (9.9 × 7.1) µm. Stieda bodies were prominent and sub-Stieda bodies were present. Two sporozoites were reflexed within each sporocyst. Nuclear 18S rRNA gene, plastid 23S rRNA gene and mitochondrial cytochrome c oxidase subunit 1 (cox1) gene were sequenced from sporulated oöcysts and compared to other molecular data of Eimeria spp. from rodent and chiropteran hosts. No sequence data in the NCBI database matched E. macyi. Phylogenetic analyses of the sequence data of the 18S rRNA and 23S rRNA genes placed E. macyi within a clade containing Eimeria spp. from rodents and basal to a clade populated by sequences derived from Eimeria spp. of rodents and bats. This account represents a new host record of E. macyi in an eastern red bat and a new geographic locality. Additionally, the cox1 sequence data of Eimeria macyi represents the first mitochondrial sequence of an Eimeria sp. in bats.

Rabbit coccidiosis is a common parasitic disease and responsible for enormous economic losses in the rabbit industry. Eimeria intestinalis, one of the highly pathogenic and common Eimeria species infecting rabbits, is considered as an indispensable species for the development of live oocyst vaccines against rabbit coccidiosis. In this study, we report the successful selection of a precocious line (EIP8) from a wild-type strain of E. intestinalis (WT) by successively collecting and propagating the early excreted progeny oocysts. The EIP8 line had a prepatent period of only 132 h compared to 204 h for the WT. Oocysts of EIP8 were notably different from those produced by the WT strain by their significantly larger size (mean length: 29.3 vs 27.6 μm and mean width 20.5 vs 19.8 μm). Examination of tissue sections prepared from EIP8-infected rabbits revealed that this precocious line undergoes only two generations of schizogony before differentiating into gametocytes by 120 h post-infection. In contrast, WT parasites undergo three generations of schizogony and gametocytes are present by 168 h post-infection. EIP8 multiplication capacity reduced by more than 35-fold and a concomitant decrease in pathogenicity was detected. Interestingly, immunization with 10 3 or 10 4 EIP8 oocysts provided sufficient protection against homologous challenge with wild-type parasites, as body weight gain of immunized and challenged rabbits was similar to that of untreated animals, as well as more than 90% reduction of oocyst output was detected in immunized and challenged animals when compared to unimmunized and challenged animals. Together, these results show that the EIP8 precocious line of E. intestinalis is an attenuated immunogenic strain and a suitable candidate for the development of a live vaccine against rabbit coccidiosis.

Green-banded broodsacs of Leucochloridium sporocysts were obtained from land snails, Succinea lauta, collected in Esashi, Esashi District, Hokkaido, Japan. The broodsacs were similar to those of L. paradoxum, which have never been found on the Japanese archipelago. Here, we compare morphological and molecular genetic characteristics of the green-banded broodsacs to those of L. paradoxum. The broodsacs described in this study and those of L. paradoxum showed three equal parts; however, band characteristics of the second parts differed. Moreover, the sequences of nuclear 18S and 28S rRNA genes of the Leucochloridium flukes were not identical with those of L. paradoxum, and the sequences of the mitochondrial cytochrome c oxidase subunit 1 gene were paraphyletic to the species. These findings suggest that the broodsacs described in this study were distinct from those of L. paradoxum. In the future, genetic analyses on adult flukes from avian hosts should be performed, upon precise identification according to morphology, in order to clarify relationships between adult flukes and sporocysts of Leucochloridium species occurring in Japan., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Coccidia of the genus Isospora, their origin, taxonomy, and host specificity have been discussed for many years. The crucial point in question being the division of the genus, based on distinct evolutionary history and the presence/absence of the Stieda body, into the genera Isospora (Eimeriidae) parasitizing mainly birds and reptiles, and Cystoisospora (Sarcocystidae) parasitizing mammals. The description of the majority of Isospora species from rodents is based solely on the oocysts found in their faeces. Some of them have been described with the presence of the Stieda body, some without it, and, simultaneously, for all the described species the molecular data are entirely lacking. This study reveals the origin of isosporan oocysts found in faeces of bank voles based on morphological analyses, phylogenetic analyses, and experimental infections. Morphological analyses showed the presence of the Stieda body complex on sporocysts. Phylogenetic analyses demonstrated close phylogenetic relationships between Isospora from bank voles and avian isosporans. Experimental inoculations of bank voles with sporulated oocysts of Isospora did not result in the production of unsporulated oocysts. Hence, these organisms should be considered pseudoparasites of the bank voles/rodents (probably originating from avian Isospora species)., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

Eimeria ammospermophili n. sp. (Apicomplexa: Eimeriidae) is described from 2 of 29 (7%) Harris's antelope squirrels Ammospermophilus harrisii Audubon & Bachman in Arizona, USA. Sporulated oöcysts of this new species are ovoidal to ellipsoidal, 24-32 × 20-25 (29.0 × 22.7) µm, with a pitted, bi-layered wall, an oöcyst residuum and, occasionally, a polar granule. Sporocysts are ellipsoidal, 10-12 × 7-9 (11.0 × 7.9) µm, with a Stieda body and sporocyst residuum; sporozoites are elongate with a spheroidal anterior refractile body and an ellipsoidal posterior refractile body. In addition, sporulated oöcysts of Eimeria vilasi Dorney, 1962 are described from A. harrisii. This is the first report on the coccidia of this host species.

In order to evaluate the abundance of oocysts in the Mezam watershed in Bamenda, Northwest Region of Cameroon, a study was carried out from January to June 2017. Samples were collected monthly from 13 stations within the watershed. The direct concentration method and the Ziehl-Neelsen technique were employed in the identification of these parasites. The physicochemical analysis showed that the water samples had a neutral pH (7.46±0.46), lowly mineralized (165.61±110.02μS/cm), moderately oxygenated (60.64 ± 17, 25%), present moderate organic pollution (2.85±2.49mg/l KMnO4). KMnO 4 ) and low levels of orthophosphate (1.8±1.88 mg/l PO 4 3- ) and nitrates (2.47±5.06 mg/l NO 3 - ). Biological analysis revealed the presence of Cryptosporidium spp. (143.98±203.35 oocysts/l), Isospora belli (88.47 ± 123.19 oocysts/l), Cyclospora cayetanensis (141.31±143.19 oocysts/l) and Sarcocystis hominis (76 ± 111.04 oocysts/l). The highest densities of these parasites were recorded at the Mufueh stream, situated in the periurban area. Meanwhile, the lowest densities were found in the urban area (Formuki, Mankon, Ayaba and Mezam streams). The dry season showed higher densities of oocysts (471.42±216.32 oocysts /l). Statistical analysis revealed a significant correlation (P ≤ 0.05) between the density of the organisms and the physico-chemical parameters such as pH, oxidability, dissolved oxygen and nitrates. Respecting basic hygienic rules as well as treating water before use would reduce the risk of contamination of the population., Competing Interests: The authors have no conflicts of interest to declare

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.)

Cryptosporidium parvum ( C. parvum) is a highly potent zoonotic pathogen, which can do significant harm to both human beings and livestock. However, existing technologies or methods are deficient for rapid on-site detection of water contaminated with C. parvum. Better detection approaches are needed to allow water management agencies to stop major breakouts of the pathogen. Herein, we present a novel detection method for cryptosporidium in a tiny drop of sample using a magnetic nanoparticle (MNP) probe combined with dark-field microscopy in 30 min. The designed MNP probes bind with high affinity to C. parvum, resulting in the formation of a golden garland-like structure under dark-field microscopy. This MNP-based dark-field counting strategy yields an amazing PCR-like sensitivity of 8 attomolar (aM) (5 pathogens in 1 μL). Importantly, the assay is very rapid (∼30 min) and is very simple to perform as it involves only one step of mixing and magnetic separation, followed by dropping on a slide for counting under dark-field microscope. By combining the advantages of the specific light-scattering characteristic of MNP probe under dark field and the selective magnetic separation ability of functionalized MNP, the proposed MNP-based dark-field enumeration method offers low cost and significant translational potential.

Concentrating diluted samples is a key step to improve detection capabilities. The wise use of scaling laws shows the advantages of working with sub-microliter-sized samples. Rapid progress in MEMS technologies has driven the design of integrated platforms performing many biochemical operations. Here we report a new concentrator device based on electro-hydrodynamic forces which can be easily integrated into electrowetting-on-dielectric (EWOD) platforms. This approach is label-free and applicable to a wide range of micro-objects. The detection and analysis of two common waterborne parasites, Cryptosporidium and Giardia, is a perfect test case due to their global health relevance. By fully controlling the interplay of the various forces acting on the micron-sized Cryptosporidium parvum and Cryptosporidium muris oocysts, we show that it is possible to concentrate them on the side of a 10 μL initial drop and then extract them efficiently from a droplet of a few hundred nanoliters. We performed a finite element modeling of the forces acting on the parasites' oocysts to optimize the electrodes' shapes. We obtained state-of-the-art concentration factors of 12 ± 0.4 times and 2 to 4 times in the sub-region of the drop and the extracted droplet, respectively, with an efficiency of 70 ± 6%. Furthermore, this device had the ability to selectively concentrate parasites of different species out of a mix. We demonstrated this by segregating C. parvum oocysts from either Giardia lamblia cysts or its related species, C. muris oocysts.

Cryptosporidiosis in an enteric infection caused by Cryptosporidium parasites and is a major cause of acute infant diarrhea in the developing world. A major bottleneck to research progress is the lack of methods to cryopreserve Cryptosporidium oocysts, thus requiring routine propagation in laboratory animals. Here, we report a method to cryopreserve C. parvum oocysts by ultra-fast cooling. Cryopreserved oocysts exhibit high viability and robust in vitro excystation, and are infectious to interferon-γ knockout mice. The course of the infection is comparable to what we observe with unfrozen oocysts. Oocyst viability and infectivity is not visibly changed after several weeks of cryogenic storage. Cryopreservation will facilitate the sharing of oocysts from well-characterized isolates and transgenic strains among different laboratories.

Understanding the importance of gametocyte density on human-to-mosquito transmission is of immediate relevance to malaria control. Previous work (Churcher et al., 2013) indicated a complex relationship between gametocyte density and mosquito infection. Here we use data from 148 feeding experiments on naturally infected gametocyte carriers to show that the relationship is much simpler and depends on both female and male parasite density. The proportion of mosquitoes infected is primarily determined by the density of female gametocytes though transmission from low gametocyte densities may be impeded by a lack of male parasites. Improved precision of gametocyte quantification simplifies the shape of the relationship with infection increasing rapidly before plateauing at higher densities. The mean number of oocysts per mosquito rises quickly with gametocyte density but continues to increase across densities examined. The work highlights the importance of measuring both female and male gametocyte density when estimating the human reservoir of infection., Competing Interests: JB, WS, DD, IM, AD, AC, WG, AM, SN, HS, HD, KL, WG, RS, Mv, IC, AT, BG, RG, RS, CD, TC, TB No competing interests declared, (© 2018, Bradley et al.)

Despite the great diversity of coccidians, to our knowledge, no coccidian infections have been described in Oecomys spp. In this context, we examined Oecomys mamorae Thomas (Rodentia: Cricetidae) from the Brazilian Pantanal for infections with enteric coccidia. Nine individuals were sampled, and one was found to be infected. The oöcysts were recovered through centrifugal flotation in sugar solution. Using morphological and morphometric features, we described a new species of Cystoisospora Frenkel, 1977. Sporulated oöcysts were ovoidal 20.0-29.1 × 16.4-23.2 (26.7 × 21.2) µm and contained two sporocysts, 12.9-19.1 × 9.4-13.9 (16.4 × 12.4) µm, each with four banana-shaped sporozoites. Polar granule and oöcyst residuum were both absent. We documented the developmental forms in the small intestine and described the histopathological lesions in the enteric tract. Our results indicate that the prevalence of Cystoisospora mamorae n. sp. in O. mamorae is low, and tissue damage in the enteric tract is mild, even in the presence of coccidian developmental stages.

A new coccidian species of the genus Isospora Schneider 1881 (Apicomplexa, Eimeriidae) is reported from a single specimen of chelid turtle, Platemys platycephala (Schneider, 1792), obtained in a flooded area of the Amazonian municipality of Altamira, in Pará State, northern Brazil. The oöcysts of Isospora platemysi n. sp. are subspheroidal, 19.0-24.0 × 22.5-18.5 (22.6 × 20.8) µm in size [length/width ratio 1.0-1.1 (1.1)], with a colourless wall, 1.0-1.9 (1.4) wide, with two smooth layers; polar granule is present, oöcyst residuum and micropyle are both absent. The sporocyst is ovoidal, 10.0-12.1 × 8.0-10.0 (11.0 × 9.1) µm [length/width ratio 1.1-1.3 (1.2), with a knob-like Stieda body. Sub-Stieda body is present, rounded irregular-trapezoidal; para-Stieda body is absent. Sporocyst residuum is dispersed and composed of granules of differing sizes. This is only the fifth record of Isospora in chelonians, and the first report of a coccidian parasite of the genus Platemys.

Three new species of coccidians (Apicomplexa: Eimeriidae) are described from eastern moles, Scalopus aquaticus (Linnaeus) from Arkansas. Oöcysts of Cyclospora duszynskii n. sp. are subspheroidal with a smooth bi-layered wall, measure 11.4 × 10.0 µm, and have a length/width (L/W) ratio of 1.1; both micropyle and oöcyst residuum are absent, but a single polar granule is present. Sporocysts are ellipsoidal and measure 7.2 × 5.4 µm, L/W 1.3; an indistinct Stieda body is present, but the sub-Stieda and para-Stieda bodies are absent and the sporocyst residuum is composed of medium to large granules of different sizes along the edge of the sporocyst. Oöcysts of Cyclospora yatesi n. sp. are subspheroidal to ovoidal with an ornate outer wall, measure 17.0 × 15.2 µm, L/W 1.1; both micropyle and oöcyst residuum are absent, but a single polar granule is present. Sporocysts are ellipsoidal and measure 9.7 × 7.3 µm, L/W 1.3; an indistinct Stieda body is present, but sub-Stieda and para-Stieda bodies are absent and the sporocyst residuum is composed of medium to large granules of different sizes along the edge of the sporocyst. Oöcysts of Eimeria paulettefordae n. sp. are ovoidal to ellipsoidal with an ornate outer wall, measure 30.0 × 25.4 µm, L/W 1.2; both micropyle and oöcyst residuum are absent, but a single polar granule is present. Sporocysts are ellipsoidal and measure 12.6 × 9.2 µm, L/W 1.4; a button-like Stieda body is present, but sub-Stieda and para-Stieda bodies are absent and the sporocyst residuum is composed of medium to large granules of different sizes along the edge of the sporocyst. These are the first coccidians described from Arkansas populations of S. aquaticus. In addition, a summary is provided on the cyclosporans and eimerians from North American talpids.

Eimeria maricopensis n. sp. (Apicomplexa: Eimeriidae) is described from 2 of 15 (13%) Arizona cotton rats Sigmodon arizonae Mearns in Arizona, USA. Sporulated oöcysts of this new species are ovoidal to ellipsoidal, 20-28 × 16-22 (24.9 × 19.2) µm, with a smooth, bi-layered wall; both micropyle and oöcyst residuum are absent, but fragmented polar granule material is present. Sporocysts are ellipsoidal, 11-14 × 6-8 (12.9 × 7.0) µm, with a Stieda body, sub-Stieda body, and sporocyst residuum; sporozoites are elongate with a spheroidal anterior refractile body and a subspheroidal posterior refractile body. In addition, sporulated oöcysts of Eimeria sigmodontis Barnard, Ernst & Dixon, 1974, Eimeria tuskegeensis Barnard, Ernst & Dixon, 1974 and Eimeria webbae Barnard, Ernst & Dixon, 1974 are described from S. arizonae. This is the first report on the coccidia of S. arizonae.