Eimeria callosciuri n. sp. (Protozoa: Eimeriidae) from Prevost's Squirrel Callosciurus prevostii Demarest, 1822 in Malaysia*

SYNOPSIS. Eimeria callosciuri n. sp. is described from Prevost's squirrel Callosciurus prevostii in Malaysia. Its oocysts are 24–31 by 20–2μ with a mean of 21.9 by 28.4μ. Schizogonic and gametogonic stages develop in the tips of the villi of the small intestine, above the nuclei of the host epithelial cells. It is the first species of Eimeria described from the rodent tribe Callosciurini.     

Phylogeny of the reptilian Eimeria: are Choleoeimeria and Acroeimeria valid generic names?

Reptiles are the animals with the most described coccidian species among all vertebrates. However, the co‐evolutionary relationships in this host–parasite system have been scarcely studied. Paperna & Landsberg (South African Journal of Zoology, 24, 1989, 345) proposed the independent evolutionary origin of the Eimeria‐like species isolated from reptiles based on morphological and developmental characteristics of their oocysts. Accordingly, they suggested the reclassification of these parasites in two new genera, Choleoeimeria and Acroeimeria. The validity of the genera proposed to classify reptilian Eimeria species remained unresolved due to the lack of species genetically characterized. In this study, we included 18S rRNA gene sequences from seven Eimeria‐like species isolated from five different lizard host families. The phylogenetic analyses confirmed the independent evolutionary origin of the Eimeria‐like species infecting lizards. Within this group, most species were placed into two monophyletic clades. One of them included the species with ellipsoidal oocysts (i.e. Choleoeimeria‐like oocysts), whereas the species with more spheroidal oocysts (i.e. Acroeimeria‐like oocysts) were included in the second one. This result supports the taxonomic validity of the genera Acroeimeria and Choleoeimeria.     

Coccidiosis in clinically asymptomatic alpaca (Vicugna pacos) crias from the Peruvian Andes

Parasites of the genus Eimeria are involved in the neonatal diarrhea complex of alpaca (Vicugna pacos) crias, and infection by Eimeria is commonly known as coccidiosis. There are limited reports of these protozoa in clinically asymptomatic crias. In this study, fecal samples from 78 clinically asymptomatic alpaca crias were analyzed to evaluate the prevalence, parasitological load, and diversity of Eimeria species. This study was conducted in the Quenamari community located in the Peruvian Andes (Marangani, Cuzco) at 4500 m above sea level. All fecal samples were examined for parasites using the quantitative McMaster and modified Stoll techniques. Microscopic examination showed the presence of Eimeria oocysts in 68 out of the 78 samples (87.18%). Among the 78 samples we found E. lamae in 67 (85.90%), E. punoensis in 49 (62.82%), E. alpacae in 42 (53.85%), E. macusaniensis in 32 (41.03%), and E. ivitaensis in four (5.13%). Regarding parasitized crias, overall there was a mean parasitological load of 43,920 oocysts per gram of feces (OPG). Eimeria lamae had the highest parasitological load (mean 206,600 OPG). These findings could be due to environmental contamination with oocysts of different Eimeria species. Additional research is needed to determine if this burden of coccidiosis could produce subclinical impacts to the health of alpaca crias.     

The Oocysts of Eimeria vermiformis sp. n. and E. papillata sp. n. (Protozoa: Eimeriidae) from the Mouse Mus musclus

SYNOPSIS. Eimeria vermiformis sp. n. and E. papillata sp. n. are described from the mouse Mus musculus. The sporulated oocysts of E. vermiformis are 18–26 by 15–21 μ (mean 23.1 by 18.4 μ); its sporocysts are 11–14 by 6–10 μ (mean 12.8 by 7.9 p). The sporulated oocysts of E. papillata are 18–26 by 16–24 μ (mean 22.4 by 19.2 μ); its sporocysts are 10–13 by 6–9 μ (mean 11.2 by 8.0 μ). A substiedal body is present in E. papillata sporocysts. Patent infections were produced in white laboratory mice with both species. Fourteen species of Eimeria have now been described from the genus Mus.     

Case study: Pathological and phylogenetic analysis of coccidiosis in two goats with heavy infection of unrecorded Eimeria sp.

Two 3-month-old goats (Capra aegagrus hircus and C. hircus coreanae) died after ataxia. In both goats, white nodules 3 mm in diameter were scattered from the duodenum to the ileum and well-raised white nodules 2–3 mm–diameter in the mucosa of the small intestine. Histopathologically, numerous mucosal polyps with coccidial oocysts were observed in the small intestine and several schizonts, macrogametocytes, microgametocytes, and macrogametes were observed in mucosal polyps in the jejunum. Based on fecal flotation tests, the oocysts morphologically resembled those of Eimeria christenseni and E. sundarbanensis; however, their sizes were different. The 18S rRNA gene and COI were phylogenetically analyzed for the molecular identification and characterization of Eimeria sp. Based on 18S rRNA gene similarity, the isolates formed an independent cluster within the related goat Eimeria clade, and the closest species were E. christenseni C2_42, E. hirci C2_99, and E. arloingi C2_119. Furthermore, these were also distinguished from other related goat Eimeria spp. in the phylogenetic tree based on the COI gene. Considering all histopathological, morphological, and phylogenetic analyses, the current study was diagnosed as fatal coccidiosis due to heavy infection with an unrecorded Eimeria species. Thus, we report in this study with caution regarding coccidiosis caused by an unrecorded Eimeria.     

Coccidia in rabbits (Oryctolagus cuniculus) in Saudi Arabia

Kasim A. A. and Al-Shawa Y. R. 1987. Coccidia in rabbits (Oryctolagus cuniculus) in Saudi Arabia. International Journal for Parasitology17: 941–944. Seven species of coccidia were identified and described for the first time in Saudi Arabia in 263 faecal samples of domestic rabbits (Oryctolagus cuniculus). A total of 73% of the specimens were positive, most of them contained 1000–10,000 oocysts per gram. Mixed infections with three Eimeria species were common. Eimeria perforans and E. media occurred most frequently, E. magna, E. coecicola and E. irresidua were less common and E. flavescens and E. intestinalis were relatively rare.     

Coccidian Parasites and Conservation Implications for the Endangered Whooping Crane (Grus americana)

While the population of endangered whooping cranes (Grus americana) has grown from 15 individuals in 1941 to an estimated 304 birds today, the population growth is not sufficient to support a down-listing of the species to threatened status. The degree to which disease may be limiting the population growth of whooping cranes is unknown. One disease of potential concern is caused by two crane-associated Eimeria species: Eimeria gruis and E. reichenowi. Unlike most species of Eimeria, which are localized to the intestinal tract, these crane-associated species may multiply systemically and cause a potentially fatal disease. Using a non-invasive sampling approach, we assessed the prevalence and phenology of Eimeria oocysts in whooping crane fecal samples collected across two winter seasons (November 2012–April 2014) at the Aransas National Wildlife Refuge along the Texas Gulf coast. We also compared the ability of microscopy and PCR to detect Eimeria in fecal samples. Across both years, 26.5% (n = 328) of fecal samples were positive for Eimeria based on microscopy. Although the sensitivity of PCR for detecting Eimeria infections seemed to be less than that of microscopy in the first year of the study (8.9% vs. 29.3%, respectively), an improved DNA extraction protocol resulted in increased sensitivity of PCR relative to microscopy in the second year of the study (27.6% and 20.8%, respectively). The proportion of positive samples did not vary significantly between years or among sampling sites. The proportion of Eimeria positive fecal samples varied with date of collection, but there was no consistent pattern of parasite shedding between the two years. We demonstrate that non-invasive fecal collections combined with PCR and DNA sequencing techniques provides a useful tool for monitoring Eimeria infection in cranes. Understanding the epidemiology of coccidiosis is important for management efforts to increase population growth of the endangered whooping crane.     

Coccidian Parasites (Apicomplexa: Eimeriidae) from Insectivores. II. Six New Species from Japanese Shrew Moles (Talpidae)1

ABSTRACT. Thirty-eight of 51 (74.5%) shrew moles collected in Japan were infected with from one to four species of Eimeria and/or Isospora including six of six Dymecodon pilirostris and 32 of 45 (71.1%) Urotrichus talpoides. Four eimerians and two isosporans were identified and all are described as new species. Sporulated oocysts of Eimeria amorphospora n. sp. were subspheroid/ellipsoid, 21.1 × 17.9(18-25 × 16-21) μm. Sporocysts were amorphous, gelatinoid envelopes 20.3 × 7.5 (17–24 × 7–9) μm. Sporozoites were enclosed together within a membrane in each sporocyst. This species was found in 9 of 45 (20%) U. talpoides. Sporulated oocysts of Eimeria gonocilia n. sp. were subspheroid/ellipsoid, 28.8 × 24.4 (25–30 × 21–28) μm; a highly ornate outer oocyst wall gave the appearance of a ciliated ball. Sporocysts ovoid, pointed at both ends, were 17.0 × 9.0 (15–19 × 7–11) μm; this species was found in 4 of 45 (8.9%) U. talpoides. Sporulated oocysts of Eimeria talpoidei n. sp. were asymmetrical ovoid, 20.6 × 13.3 (18–23 × 12–15) μm, with sporocysts lacrimiform, 12.0 × 5.8 (10–14 × 5–7) μm. This species was found in 7 of 45 (15.6%) U. talpoides. Sporulated oocysts of Eimeria honshuensis n. sp. were ellipsoid, 15.5 × 11.4 (13–18 × 10–13) μm, with sporocysts ovoid, 9.1 × 5.2 (8–10 × 4–6) μm. This species was found in 10 of 45 (22.2%) U. talpoides and in 5 of 6 (83.3%) D. pilirostris. Sporulated oocysts of Isospora dymecodi n. sp. were subspheroid/ellipsoid, 15.8 × 12.6 (13–17 × 11–13) μm, with sporocysts ellipsoid, 10.9 × 6.9 (10–13 × 6–8). This species was found in six of six D. pilirostris. Sporulated oocysts of Isospora urotrichi n. sp. were spheroid/subspheroid, 13.4 × 12.4 (11–16 × 9–14) μm, with sporocysts ovoid, 9.2 × 6.3 (8–11 × 5–7) μm. This species was found in 27 of 45 (60%) U. talpoides. Only 14 of 38 (36.8%) infected hosts (one D. pilirostris, 13 U. talpoides) were seen to be naturally infected with only one coccidian species when sampled.     

Pleistocene Carnivora (Mammalia) from Tighennif (Ternifine), Algeria

The site of Tighennif (= Ternifine) in Northern Algeria, well-known for its Homo mauritanicus (= Homo rhodesiensis?) remains, and probably dating to the late Calabrian, yielded a large assemblage of terrestrial carnivores. Some are identical or probably identical with extant species: Crocuta crocuta and Hyaena hyaena (Hyaenidae), Felis silvestris (Felidae), Mellivora capensis and Poecilictis cf. libyca (Mustelidae), and Vulpes cf. rueppelli (Canidae). In addition, among felids there is an unidentified leopard-like form; a smaller, more common species assigned to Lynx sp. (a genus quite rare in Africa) but which is certainly different from modern forms, an Homotherium that seems to be the last occurrence of the machairodonts in Africa, and a Panthera aff. leo, which is unfortunately too poorly known to be named. Rare bears do not display all derived features of later North African U. bibersoni. Among canids, the Nyctereutes-like jackal Lupulella mohibi is an endemic North African form known until the late middle Pleistocene, and the hunting dog Lycaon magnus is also clearly distinct from the modern species. A single new species is described, Enhydrictis hoffstetteri, a large, otter-like member of the Mustelidae, of a genus that was previously unknown from Africa, and certainly testifies to North–South dispersal across the Mediterranean at some time during the early Pleistocene.     

A new species of Eimeria Schneider, 1875 (Apicomplexa: Eimeriidae) from the dusky rice rat Melanomys caliginosus (Tomes) (Mammalia: Rodentia) in Costa Rica

We collected faecal samples from 24 dusky rice rats, Melanomys caliginosus (Tomes) (Rodentia: Cricetidae: Sigmodontinae), in a Biological Reserve in Costa Rica, and found three (12.5%) to be infected with a species of Eimeria Schneider, 1875, which we describe here as new. Sporulated oöcysts of Eimeria caliginosa n. sp. are almost spheroidal and measure 16–21 × 17–20 (mean 19.6 × 18.2) μm; micropyle, oöcyst residuum and polar granule are absent. Sporocysts are ovoidal, 9–13 × 6–8 (mean 11.2 × 6.7) μm, with small Stieda and sub-Stieda bodies present, but a para-Stieda body is absent; the sporocyst residuum is a compact mass of c.11–15 granules, c.5 μm wide. Sporozoites are crescent-shaped, 5–8 × 2–3 (mean 6.8 × 2.4) μm. This is the third species of Eimeria described from the genus Melanomys Thomas.     

Eimeria ladronensis n. sp. and E. albigulae (Apicomplexa: Eimeriidae) from the Woodrat,Neotoma albigula (Rodentia: Cricetidae)1

Of 50 white-throated woodrats (Neotoma albigula) collected from Socorro Co., New Mexico, 21 (42%) had eimerian oocysts in their feces when examined. Of the 21 Neotoma found positive for Eimeria, 19 (90%) harbored a single eimerian species at time of examination. Eimeria albigulae Levine, Ivens & Kruidenier, 1957, was found in 18 (86%), and E. ladronensis n. sp. was found in five (24%) infected woodrats. Sporulated oocysts of E. ladronensis are ellipsoidal, 19–25 × 13–15 (21.4 ± 1.3 × 14.1 ± 1.1) μm, have a smooth wall and one or two polar granules, but lack a micropyle and an oocyst residuum. Sporocysts are tapered at one end, 7–10 × 6–7 (8.5 ± 0.7 × 6.5 ± 0.3) μm, and have a Stieda body and sporocyst residuum, but no substieda body. Prepatent periods for E. albigulae and E. ladronensis n. sp. are 5–6 and 8–9 days, respectively; patent periods are 7–18 and approximately 11 days, respectively.     

Eimeria atheridis n. sp. (Apicomplexa: Eimeriidae), a new coccidium from the western bush viper Atheris chlorechis (Pel, 1851) from tropical Africa

Coprological examination of nine bush vipers Atheris chlorechis imported from Ghana revealed the presence of a new coccidian species belonging to Eimeria Schneider, 1875. Thin walled oöcysts of Eimeria atheridisn. sp. are spherical to slightly subspherical, 22.8 (19–26) × 22.5 (19–25) μm, without micropyle, polar granule and oöcyst residuum. Sporocysts are elongately ellipsoidal, 17.1 (15–19) × 7.5 (6–8) μm, with a dome like, relatively flat Stieda body. Sporozoites possess two refractile bodies and distinct transversal striation. Based on the presence of a Stieda body the species described herein clearly belongs to the Eimeria (sensu stricto).     

Eimeria indianensis sp. n. and an Isospora sp. from the Opossum Didelphis virginiana (Kerr)*

Two of 15 road-killed opossums examined for coccidia were found to be infected with a hitherto undescribed species of Eimeria, herein named Eimeria indianensis . The oocysts were spherical (63%) or slightly subspherical (37%) with a double-layered wall. The outer layer was ～1.5 μm thick, yellowish, striated, and appeared rough and pitted on the surface. A micropyle was absent. The spherical oocysts were 16.3 (13–18) μm in diameter; the subspherical ones, 17.6 (15–18) × 16.4 (14–17) μm. The sporocysts measured 9.1 (8–10) × 6.2 (6–7) μm and contained a granular residuum. The sporozoites were elongate, measuring 13.4 (13–15) × 1.8 (1.6-2.0) μm; no refractile globules were seen. The prepatent period was 10 days and the patent period ranged from 9–15 days. A few oocysts of an Isospora sp. were present in one opossum. It was not possible to confirm whether they were specifically of the opossum or of spurious origin.     

Evaluation of Cryptosporidium parvum Genotyping Techniques

We evaluated the specificity and sensitivity of 11 previously described species differentiation and genotyping PCR protocols for detection of Cryptosporidium parasites. Genomic DNA from three species of Cryptosporidium parasites (genotype 1 and genotype 2 of C. parvum, C. muris, and C. serpentis), two Eimeria species (E. neischulzi and E. papillata), and Giardia duodenalis were used to evaluate the specificity of primers. Furthermore, the sensitivity of the genotyping primers was tested by using genomic DNA isolated from known numbers of oocysts obtained from a genotype 2 C. parvum isolate. PCR amplification was repeated at least three times with all of the primer pairs. Of the 11 protocols studied, 10 amplified C. parvum genotypes 1 and 2, and the expected fragment sizes were obtained. Our results indicate that two species-differentiating protocols are not Cryptosporidium specific, as the primers used in these protocols also amplified the DNA of Eimeria species. The sensitivity studies revealed that two nested PCR-restriction fragment length polymorphism (RFLP) protocols based on the small-subunit rRNA and dihydrofolate reductase genes are more sensitive than single-round PCR or PCR-RFLP protocols.     

Eimeria cicaki sp. n. and Isospora thavari sp. n. from the House Lizard Gehyra mutilata Boulenger in Malaysia*

SYNOPSIS. Oocysts and endogenous stages of new species of Eimeria and Isospora from the house lizard, Gehyra mutilata, are described. The ellipsoid to subspherical 2-layered oocysts of E. cicaki averaged 24.0 × 21.0 μm. Polar granules are present. Micropyle and oocyst residuum are absent. Ellipsoid sporocysts average 12.2 × 9.0 μm. A sporocyst residuum is present, but the Stieda body is absent. Endogenous stages are in epithelial cells of the small intestine. The subspherical single-layered oocysts of I. thavari average 23.8 × 22.8 μm. The polar granule is present; micropyle and oocyst residuum are absent. Ellipsoid sporocysts average 12.8 × 9.4 μm. Stieda body and sporocyst residuum are present. There are endogenous stages in epithelial cells of the small intestine.     

Ossicaulis yunnanensis sp. nov. (Lyophyllaceae,Agaricales) from southwestern China

Ossicaulis is a small genus in the family Lyophyllaceae. Two known species, O. lachnopus, and O. lignatilis, are distributed in north temperate regions. The third taxon, O. yunnanensis sp. nov., is described from the alpine belt of subtropics of southwestern China, and this genus is also reported for the first time from China. Morphologically, the new species is characterized by its whitish basidiomata and very small basidiospores. Molecular analyses from the nuclear rDNA internal transcribed spacer region (ITS) show that the subtropical alpine species is distinct from hitherto known Ossicaulis species and has a close relationship to O. lachnopus.     

Optimized excystation protocol for ruminant Eimeria bovis- and Eimeria arloingi-sporulated oocysts and first 3D holotomographic microscopy analysis of differing sporozoite egress

Successful excystation of sporulated Eimeria spp. oocysts is an important step to acquire large numbers of viable sporozoites for molecular, biochemical, immunological and in vitro experiments for detailed studies on complex host cell-parasite interactions. An improved method for excystation of sporulated oocysts and collection of infective E. bovis- and E. arloingi-sporozoites is here described. Eimeria spp. oocysts were treated for at least 20 h with sterile 0.02 M _L-cysteine HCl/0.2 M NaHCO₃ solution at 37 °C in 100% CO₂ atmosphere. The last oocyst treatment was performed with a 0.4% trypsin 8% sterile bovine bile excystation solution, which disrupted oocyst walls with consequent activation of sporozoites within oocyst circumplasm, thereby releasing up to 90% of sporozoites in approximately 2 h of incubation (37 °C) with a 1:3 (oocysts:sporozoites) ratio. Free-released sporozoites were filtered in order to remove rests of oocysts, sporocysts and non-sporulated oocysts. Furthermore, live cell imaging 3D holotomographic microscopy (Nanolive®) analysis allowed visualization of differing sporozoite egress strategies. Sporozoites of both species were up to 99% viable, highly motile, capable of active host cell invasion and further development into trophozoite- as well as macroment-development in primary bovine umbilical vein endothelial cells (BUVEC). Sporozoites obtained by this new excystation protocol were cleaner at the time point of exposure of BUVEC monolayers and thus benefiting from the non-activation status of these highly immunocompetent cells through debris. Alongside, this protocol improved former described methods by being is less expensive, faster, accessible for all labs with minimum equipment, and without requirement of neither expensive buffer solutions nor sophisticated instruments such as ultracentrifuges.     

Eimeria acervulina,E. brunetti,E. maxima, and E. necatrix: Low doses of oocysts to immunize young chickens

Resistance to reinfection varied with the species of Eimeria and with the number of oocysts in the inoculum. Chickens immunized with doses of 20,000 and 80,000 oocysts of E. acervulina, 312 and 1250 oocysts of E. brunetti or E. necatrix, or 1250 and 5000 oocysts of E. maxima at 2 and 4 weeks of age, respectively, were almost completely immune to a challenge dose at 6 weeks of age. Resistance was slightly less in chickens immunized with 1250 and 5000 oocysts of E. acervulina or 312 and 1250 oocysts of E. maxima. Birds given three immunizing infections of 1250, 5000, and 20,000 oocysts of E. maxima were completely immune 8 weeks after the last dose. Resistance was slightly less in birds immunized with similar doses of E. brunetti or E. necatrix. Doses of 20,000, 80,000, and 320,000 oocysts appeared necessary to confer a high level of immunity to E. acervulina. More than three low doses of oocysts appear necessary to induce a complete and enduring immunity against a high challenge for E. acervulina, E. brunetti, and E. necatrix. Higher immunizing doses would not be satisfactory due to the pathogenic effects of the coccidia after the initial infection.     

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

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