Molecular phylogeny of the other tissue coccidia: Lankesterella and Caryospora

Nearly complete sequences were obtained from the 18S rDNA genes of Eimeria falciformis (the type species of the genus), Caryospora bigenetica, and Lankesterella minima. Two clones of the rDNA gene from C. higenetica varied slightly in primary structure. Parsimony-based and maximum likelihood phylogenetic reconstructions with a number of other apicomplexan taxa support 2 major clades within the Eucoccidiorida, i.e., the isosporoid coccidia (consisting of Toxoplasma, Neospora, Isospora [in part], and Sarcocystis spp.) and a second clade containing Lankesterella and Caryospora spp., as well as the eimeriid coccidia (Cyclospora, Isospora [in part], and Eimeria spp.). Our observations suggest that Caryospora spp. may not belong in the family Eimeriidae but rather may be allied with the family Lankesterellidae with which they share molecular and life history similarities. This may be a third lineage of coccidian parasites that has independently evolved a unique heteroxenous transmission strategy.     

Detection and differentiation of coccidian oocysts by real-time PCR and melting curve analysis

Rapid and reliable detection and identification of coccidian oocysts are essential for animal health and foodborne disease outbreak investigations. Traditional microscopy and morphological techniques can identify large and unique oocysts, but they are often subjective and require parasitological expertise. The objective of this study was to develop a real-time quantitative PCR (qPCR) assay using melting curve analysis (MCA) to detect, differentiate, and identify DNA from coccidian species of animal health, zoonotic, and food safety concern. A universal coccidia primer cocktail was designed and employed to amplify DNA from Cryptosporidium parvum, Toxoplasma gondii, Cyclospora cayetanensis, and several species of Eimeria, Sarcocystis, and Isospora using qPCR with SYBR Green detection. MCA was performed following amplification, and melting temperatures (T(m)) were determined for each species based on multiple replicates. A standard curve was constructed from DNA of serial dilutions of T. gondii oocysts to estimate assay sensitivity. The qPCR assay consistently detected DNA from as few as 10 T. gondii oocysts. T(m) data analysis showed that C. cayetanensis, C. parvum, Cryptosporidium muris, T. gondii, Eimeria bovis, Eimeria acervulina, Isospora suis, and Sarcocystis cruzi could each be identified by unique melting curves and could be differentiated based on T(m). DNA of coccidian oocysts in fecal, food, or clinical diagnostic samples could be sensitively detected, reliably differentiated, and identified using qPCR with MCA. This assay may also be used to detect other life-cycle stages of coccidia in tissues, fluids, and other matrices. MCA studies on multiple isolates of each species will further validate the assay and support its application as a routine parasitology screening tool.     

Coccidian parasites (Apicomplexa: Eimeriidae) from insectivores. VII. Six new species from the hairy-tailed mole, Parascalops breweri

Sixteen hairy-tailed moles, Parascalops breweri, collected from the northeastern U.S.A. were examined for coccidian oocysts; all were infected with multiple species of coccidia and 3 genera were represented. Two cyclosporans, 2 eimerians, and 2 isosporans are described as new species. Sporulated oocysts of Cyclospora ashtabulensis n. sp. are subspheroid to ellipsoid, 18 X 14 (14-23 X 11-19) microns, and sporocysts are ovoid, 12 X 7 (8-14 X 5-9) microns; C. ashtabulensis was found in 7 of 16 (44%) moles. Sporulated oocysts of Cyclospora parascalopi n. sp. are spheroid, 17 X 14 (13-20 X 11-20) microns, and sporocysts are ovoid, 11 X 7 (8-14 X 5-8) microns; C. parascalopi was found in 8 of 16 (50%) moles. Sporulated oocysts of Eimeria aethiospora n. sp. are subspheroid to ellipsoid, 19 X 13 (15-24 X 10-16) microns, and sporocysts are ovoid, 11 X 6 (8-13 X 4-7) microns; E. aethiospora was found in 4 of 16 (25%) moles. Sporulated oocysts of Eimeria titthus n. sp. are subspheroid, 16 X 14 (13-19 X 11-17) microns, and sporocysts are ellipsoid, 11 X 6 (9-13 X 4-7) microns; E. titthus was found in 4 of 16 (25%) moles. Sporulated oocysts of Isospora ashtabulensis n. sp. are ellipsoid, 20 X 14 (16-24 X 10-18) microns, and sporocysts are ovoid, 10 X 7 (7-14 X 5-10) microns; I. ashtabulensis was found in 5 of 16 (31%) moles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Prevalence of coccidia parasites (Protozoa) in red squirrels ( Sciurus vulgaris): effects of host phenotype and environmental factors

We investigated the relative importance of environmental factors versus host phenotype in determining parasite prevalence in Eurasian red squirrels ( Sciurus vulgaris). One hundred and forty-three fecal samples of 116 different squirrels collected in 2000 and 2001 from five study areas in the Italian Alps, were examined for intestinal protozoans. Two species of Eimeria were present with a medium to high prevalence in both years and in all areas, while two other species were rare, occurring only in some areas and not in all years. Cryptosporidium parvum had a high prevalence in the two study areas of the Western Alps, while in the three areas of the Central Alps it was recorded only once. The prevalence of Eimeria sciurorum and C. parvum fluctuated in parallel with squirrel density, suggesting a possible correlation between the presence of these protozoans and host density. A gender effect on E. sciurorum prevalence at low density could be explained by different space use patterns and social organization of males and females. C. parvum occurred more frequently in young squirrels, suggesting an acquired immunity in adults, but age-related susceptibility was not found for eimerian species. The coccidian community was more similar within than between regions, and study area and year were key parameters in predicting coccidia infection. There was no evidence of competition between coccidian species, but one positive interaction between E. sciurorum and E. andrewsi was observed. Our results suggest that the effects of geographic region, area features, and year effects probably related to fluctuations in host population density, were more important than individual phenotypic host characteristics in structuring the coccidian assemblage and determining levels of parasite prevalence in red squirrel populations.     

Coccidian Parasites (Apicomplexa: Eimeriidae) from Insectivores. IV. Four New Species in Talpa europaea from England1

ABSTRACT. Moles from England were examined for coccidian oocysts and all 64 Talpa europaea were infected; of 64 infected hosts, 56 (88%) had multiple infections representing two to six coccidian species when examined. Oocysts in 31 of the 64 samples remained unsporulated. Three eimerians and one isosporan were studied from the 33 fecal samples that had sporulated oocysts and these are described as new species; Cyclospora talpae Pellérdy & Tanyi, 1968, and Isospora sofiae (Golemansky, 1978) Levine & Ivens, 1979, are redescribed; and Cyclospora sp., similar to C. talpae, is discussed. Sporulated oocysts of C. talpae are ellipsoidal, 14.3 × 9.6 (12–19 × 6–13) μm with sporocysts ovoid, 9.4 × 5.7 (6–13 × 4–8) μm; it was found in 21 of the 33 (63.6%) sporulated samples. Sporulated oocysts of Cyclospora sp. are subspheroidal to ellipsoidal, 12.5 × 8.9 (10–14 × 6–12) μm with sporocysts ovoid, 8.6 × 5.3 (6–10 × 4–6) μm; it was found in 21 of the 33 (63.6%) sporulated samples. Sporulated oocysts of Eimeria avonensis n. sp. are elongate-ellipsoidal, 15.0 × 9.6 (13–20 × 7–12) μm with sporocysts ovoid, 6.6 × 3.6 (5–9 × 3–7) μm; it was found in 15 of the 33 (45.5%) sporulated samples. Sporulated oocysts of Eimeria berea n. sp. are subspheroidal, 12.1 × 10.5 (10–15 × 8–14) μm with sporocysts ovoid, 6.3 × 3.9 (5–10 × 2–5) μm; it was found in 8 of the 33 (24.2%) sporulated samples. Sporulated oocysts of Eimeria globula n. sp. are spheroidal, 20.9 × 19.9 (19–24 × 17–21) μm with sporocysts elongate-ovoid, 11.5 × 6.9 (9–16 × 6–9) μm; it was found in 3 of the 33 (9.1%) sporulated samples. Sporulated oocysts of Isospora sporopointaea n. sp. are subellipsoidal to ellipsoidal, 17.1 × 11.4 (13–21 × 8–14) μm with sporocysts ellipsoidal with both ends pointed, 11.9 × 5.9 (9–16 × 4–8) μm; it was found in 27 of the 33 (81.8%) sporulated samples. Sporulated oocysts of I. sofiae are spheroidal to subspheroidal, 12.2 × 11.0 (9–16 × 8–15) μm with sporocysts ovoid, 9.1 × 5.2 (6–13 × 3–8) μm; it was found in 25 of the 33 (75.8%) sporulated samples. To date, the coccidian parasites of talpids include two cyclosporans, 12 eimerians, and six isosporans, exclusive of the four new species described here.     

Five new species of Coccidia (Apicomplexa: Eimeriidae) from colubrid snakes of Ecuador.

Fecal samples from 11 colubrid snakes, representing 10 species, collected in Ecuador during October 1994 were examined for coccidian parasites. Feces of 4 individuals, representing 4 host species, contained coccidian oocysts. Three species of Eimeria and 2 species of Isospora were observed and are described here as new. Oocysts of both Eimeria and Isospora were found in the feces of a slug-eating snake, Dipsas vermiculata. Sporulated oocysts of the Eimeria sp. are spheroid to subspheroid, 16.7 by 16.6 microm (14-18 by 14-18 microm) and those of the Isospora sp. are spheroid and 15.0 microm (13-18 microm) in diameter. Imantodes cenchoa, the common bluntheaded treesnake, was infected with a species of Eimeria. These sporulated oocysts are ellipsoid, 23.3 by 16.2 microm (25-21 by 15-17 microm). Sporulated eimerian oocysts from Leptodeira annulata, the southern cat-eyed snake, are subspheroid, 22.5 by 18.8 microm (19-26 by 17-21 microm). Feces of a juvenile Imantodes lentiferus, the bluntheaded vine snake, contained ovoid to ellipsoid isosporan oocysts, which measured 21.6 by 15.0 microm (20-23 by 14-16 microm) when sporulated.     

Coccidian parasites (Apicomplexa: Eimeriidae) from two species of caimans, Caiman yacare daudin and Caiman latirostris daudin (Alligatoridae), from Paraguay

From October 1986 to January 1987, feces from 119 Caiman yacare and 12 Caiman latirostris were collected in Paraguay and later examined for coccidian oocysts; 69 of 119 (58%) samples from C. yacare and 3 of 12 (25%) samples from C. latirostris contained coccidian oocysts. Two eimerians infected C. yacare and both are described as new species. Sporulated oocysts of Eimeria paraguayensis n. sp. are ellipsoid, 34.0 x 23.6 (26-38 x 20-29) microns with sporocysts ovoid, 14.0 x 7.1 (10-19 x 6-10) microns. Sporulated oocysts of Eimeria caimani n. sp. are spheroid, 22.4 (19-29) microns with sporocysts ovoidal, 12.9 x 6.5 (8-17 x 5-8) microns. Isospora jacarei infected C. latirostris and is redescribed. Sporulated oocysts of I. jacarei are sub-spheroid, 13.2 x 12.1 (10-18 x 10-15) microns with sporocysts ellipsoid, 10.4 x 5.8 (7-13 x 4-11) microns. To date, members of the Eimeriidae found in Crocodylia include 5 species of Eimeria and 2 of Isospora including the new species described here.     

Eimeria melogale n. sp. (Apicomplexa: Eimeriidae) in the Javan ferret-badger (Melogale orientalis)

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.     

Experimental Isospora canis and Isospora felis Infection in Mice,Cats, and Dogs*

SYNOPSIS. Two 6-month-old gnotobiotic dogs and 4 five-day-old dogs became infected but did not shed oocysts within 15 days after ingesting the feline coccidian, Isospora felis. Infection of the dogs was evidenced by the shedding of I. felis oocysts by cats consuming extra-intestinal organs of dogs fed I. felis. Likewise, cats became infected with the canine coccidian, Isospora canis, without producing oocysts. Dogs also became infected after ingesting mice previously fed I. canis oocysts. The prcpatent period for I. canis was slightly shorter in dogs fed infected mice (8–9 days) than in those fed oocysts (9–11 days).     

Coccidian parasites (Apicomplexa: Eimeriidae) from insectivores. IV. Four new species in Talpa europaea from England

Moles from England were examined for coccidian oocysts and all 64 Talpa europaea were infected; of 64 infected hosts, 56 (88%) had multiple infections representing two to six coccidian species when examined. Oocysts in 31 of the 64 samples remainedunsporulated. Three eimerians and one isosporan were studied from the 33 fecal samples that had sporulated oocysts and these are described as new species; Cyclospora talpae Pellérdy & Tanyi, 1968, and Isospora sofiae (Golemansky, 1978) Levine & Ivens, 1979, are redescribed; and Cyclospora sp., similar to C. talpae, is discussed. Sporulated oocysts of C. talpae are ellipsoidal, 14.3 X 9.6 (12-19 X 6-13) microns with sporocysts ovoid, 9.4 X 5.7 (6-13 X 4-8) microns; it was found in 21 of the 33 (63.6%) sporulated samples. Sporulated oocysts of Cyclospora sp. are subspheroidal to ellipsoidal, 12.5 X 8.9 (10-14 X 6-12) microns with sporocysts ovoid, 8.6 X 5.3 (6-10 X 4-6) microns; it was found in 21 of the 33 (63.6%) sporulated samples. Sporulated oocysts of Eimeria avonensis n. sp. are elongate-ellipsoidal, 15.0 X 9.6 (13-20 X 7-12) microns with sporocysts ovoid, 6.6 X 3.6 (5-9 X 3-7) microns; it was found in 15 of the 33 (45.5%) sporulated samples. Sporulated oocysts of Eimeria berea n. sp. are subspheroidal, 12.1 X 10.5 (10-15 X 8-14) microns with sporocysts ovoid, 6.3 X 3.9 (5-10 X 2-5) microns; it was found in 8 of the 33 (24.2%) sporulated samples.(ABSTRACT TRUNCATED AT 250 WORDS)     

Coccidia (Apicomplexa) from heteromyid rodents in the southwestern United States, Baja California, and northern Mexico with three new species from Chaetodipus hispidus

Fecal samples from 223 heteromyid rodents of 4 genera and 13 species were collected from California, New Mexico, and Texas and from Baja California Norte and Sonora, Mexico. Of these, 84 (38%) were infected with coccidian oocysts; 72 of 84 (86%) infected animals had only 1 species of coccidian. Eleven species of coccidia were identified including 1 cyclosporan and 10 eimerians; the cyclosporan and 2 of the eimerians are described as new species. Sporulated oocysts of Cyclospora angimurinensis n. sp. were subspheroidal, 21.9 x 19.3 (19-24 x 16-22) microns, with sporocysts lemon-shaped, 11.9 x 9.5 (9-15 x 8-11) microns; it was found in 1 of 20 (4%) Chaetodipus hispidus. Sporulated oocysts of Eimeria chaetodipi n. sp. were subspheroidal, 16.7 x 14.6 (13-19.5 x 12-17) microns, with sporocysts ovoidal, 8.7 x 6.6 (7.5-10.5 x 5-7.5) microns; it was found in 3 of 20 (15%) C. hispidus. Sporulated oocysts of Eimeria hispidensis n. sp. were subspheroidal, 20.5 x 17.4 (17-23 x 14-21) microns, with sporocysts lemon-shaped, 9.3 x 7.2 (7.5-10.5 x 5-9) microns; it was found in 4 of 20 (20%) C. hispidus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Coccidian Parasites (Apicomplexa: Eimeriidae) from Insectivores. VI. Six New Species from the Eastern Mole,Scalopus aquaticus1

ABSTRACT Thirteen eastern moles, Scalopus aquaticus, collected in West Texas were examined for coccidian oocysts; 11 (85%) were infected and eight (73%) of these had multiple infections representing two or more species. One cyclosporan, three eimerians, and two isosporans were studied and all are described as new species. Sporulated oocysts of Cyclospora megacephali n. sp. were subspheroidal, 18.5 × 15.7 (14–21 × 12–18) μm; they had sporocysts pointed at one end with Stieda bodies nearly as wide as the sporocysts themselves, and were 15.0 × 7.2 (11–17 × 6–9) μm; C. megacephali was found in four (31%) hosts. Sporulated oocysts of Eimeria scalopi n. sp. were spheroidal to subspheroidal, 13.6 × 12.6 (11–17 × 11–15) μm with sporocysts lemon-shaped, 8.7 × 5.5 (7–10 × 4–7) μm; it was found in six (46%) hosts. Sporulated oocysts of Eimeria aquatici n. sp. were asymmetrically ellipsoidal, 17.0 × 10.6 (14–20 × 9–14) μm, with sporocysts elongately ovoidal, 9.0 × 5.2 (8–11 × 4–6) μm; it was found in two (15%) hosts. Sporulated oocysts of Eimeria motleiensis n. sp. were subspheroidal, 17.0 × 15.3 (15–20 × 13–18) μm with sporocysts ovoidal, 10.7 × 6.8 (10–13 × 6–8) μm; it was found in seven (54%) hosts. Sporulated oocysts of Isospora motleiensis n. sp. were spheroidal to subspheroidal, 13.6 × 12.0 (10–17 × 8–15) μm with sporocysts broadly ovoidal, 9.5 × 6.7 (7–11 × 4–8) μm; it was found in nine (69%) hosts. Sporulated oocysts of Isospora aquatici n. sp. were subspheroidal, 20.9 × 18.4 (15–24 × 13–21) μm with sporocysts ellipsoidal, 11.8 × 9.0 (9–14 × 7–11) μm; it was found in two (15%) hosts.     

Attempted Cross-Transmission of Coccidia Between Sheep and Goats and Description of Eimeria ovinoidalis sp. n.

SYNOPSIS.
Attempted infection of 2 young lambs with oocysts of Eimeria christenseni from a goat was unsuccessful. Negative results were obtained also when young kids were fed oocysts of Eimeria ninakohlyakimovae from sheep. There was no difficulty in infecting lambs with the sheep coccidium resembling E. ninakohlyakimovae nor goats with the goat coccidium E. christenseni. Oocysts from the goat measured 38.4 × 26.7 m, but were easily distinguished from Eimeria ahsata from the sheep by sporocyst size and shape, and from Eimeria ovina by oocyst size. Eimeria ninakohlyakimovae -like oocysts from sheep averaged 23.0 ×18.2 m and were morphologically indistinguishable from previously reported goat coccidia.
Since no cross infections of sheep and goats could be accomplished with oocysts of Eimeria sp. characteristic of one or the other host, I concluded that sheep coccidia previously known as E. ninakohlyakimovae are distinct from morphologically similar goat coccidia and therefore constitute a separate species. Since the name E. ninakohlyakimovae was first used for coccidia from the goat, the sheep coccidium is renamed Eimeria ovinoidalis with oocyst structure and endogenous stages similar to those previously described from the sheep.     

Eimeria and Klossia spp. (Protozoa: Sporozoa) from Wild Mammals in Borneo*

SYNOPSIS. During a survey of parasites of Sabah mammals, coccidia oocysts were found in 9 of 22 host species examined. Sporulated oocysts of Klossia sp. from Rattus whiteheadi are described. New geographic records are reported for Eimeria callosciuri, Eimeria sabani, and Eimeria tupaiae. New host and geographic records are reported for Eimeria separata from Rattus cremoriventer, for E. tupaiae and Eimeria ferruginea from Tupaia tana, for Eimeria nieschulzi from Rattus muelleri, and for E. sabani and Klossia sp. from R. whiteheadi.     

Eimeria papillata: upregulation of specific miRNA-species in the mouse jejunum

Increasing evidence indicates miRNAs as critical regulators of gene expression, but little information is available for miRNAs in intestinal diseases. Here, we investigated intestinal infections of male Balb/c mice with the coccidian parasite Eimeria papillata. On day 4 after oral infection, mice were shedding 3150 ± 430 oocysts per gram feces. This was associated with a low inflammatory response of the jejunum of mice evidenced by histology, non-response of IL-1β mRNA, even slight downregulation of IL-6 mRNA, only slight increases in iNOS mRNA, nitrate/nitrate, malondialdehyde, and a small decrease in glutathione, respectively. Only IFNγ mRNA was strongly induced. Using miRNA microarray technology, there were significantly upregulated the four miRNA species miR-1959, MCMV-miR-M23-1-5P, miR-203, and miR-21 out of 634 miRNAs, which was also confirmed by quantitative RT-PCR. Our data provide evidence that E. papillata parasites are able to induce specific miRNA species in their host target organ.     

Attempted cross-transmission of coccidia between sheep and goats and description of Eimeria ovinoidalis sp. n.

Attempted infection of 2 young lambs with oocysts of Eimeria christenseni from a goat was unsuccessful. Negative results were obtained also when young kids were fed oocysts of Eimeria ninakohlyakimovae from sheep. There was no difficulty in infecting lambs with the sheep coccidium resembling E. ninakohlyakimovae nor goats with the goat coccidium E. christenseni. Oocysts from the goat measured 38.4 X 26.7 microns, but were easily distinguished from Eimeria ahsata from the sheep by sporocyst size and shape, and from Eimeria ovina by oocyst size. Eimeria ninakohlyakimovae-like oocysts from sheep averaged 23.0 X 18.2 microns and were morphologically indistinguishable from previously reported goat coccidia. Since no cross infections of sheep and goats could be accomplished with oocysts of Eimeria sp. characteristic of one or the other host, I concluded that sheep coccidia previously known as E. ninakohlykimovae are distinct from morphologically similar goat coccidia and therefore constitute a separate species. Since the name E. ninakohlyakimovae was first used for coccidia from the goat, the sheep coccidium is renamed Eimeria ovinoidalis with oocyst structure and endogenous stages similar to those previously described from the sheep.     

Virus-like particles in Eimeria tenella are associated with multiple RNA segments

Total nucleic acids from sporulated oocysts of Eimeria tenella isolated from Changchun in China were found to contain three extrachromosomal double-stranded RNA segments (dsRNAs) of 1.4, 2.4 and 3.6 kb in sizes. These RNAs were resistant to RNase A digestion under high salt concentration (0.3 M NaCl). RNA-dependent RNA polymerase (RDRP) activity was detected in crude extracts of E. tenella sporulated oocysts containing these nucleic acid species. Virus-like particles (VLPs) were shown to have a diameter of approximately 38 nm under Electron Microscopy (EM) after purification by sucrose density gradient centrifugation. In keeping with the nomenclature generally adopted for protozoan viruses, we have named this isolate as E. tenella virus (ETV) which is the first virus isolated from E. tenella.     

Coccidia of the domestic goat (Capra hircus) in Saudi Arabia.

Faeces of 228 domestic goats (Capra hircus) from the central region of Saudi Arabia were examined for the presence of coccidian oocysts. Ten species of coccidia were identified and described. A total of 90.3% of the specimens were positive, most of them contained 100-1000 oocysts per g of faecal sample. Kids less than 1 year old had higher oocyst counts than goatlings or adult goats. Mixed infections with three to five species were found in 69.7% of the specimens and six to eight species were found in 10.1%. Eimeria arloingi and E. hirci were most prevalent. E. alijevi, E. ninakohlyakimovae, E. caprina, E. christenseni and E. apsheronica were less common. E. jolchijevi, E. caprovina and E. punctata were relatively rare.