Analysis of Coccidian Oocyst Populations by Means of Flow Cytometry

Flow cytometry was employed as a tool to analyze and characterize batches of oocysts from laboratory and field isolates of Eimeria spp. from chickens and to propagate sub-populations of batches of oocysts. Oocyst batches were cleaned of debris by a combination of salt flotation, washing and treatment with dilute sodium hypochlorite (1.5% aqueous). Oocyst size and shape were registered by forward-angle light scatter with the argon laser excitation set at 488 nm at 300 mW. Sub-populations of oocysts were collected by map gating and used for microscopy or for propagation. The profile of particle size was characteristic for each species. Propagation of sub-populations of oocysts of specified sizes resulted in cultures of coccidia that were pure species or nearly pure species. The small size of E. mills caused difficulty in separation from the remaining fine debris. This technique was useful for studying the variation in oocyst size within populations and characterization of field isolates of mixed species. Propagation of pure species from mixed isolates by bit-map gating had the same limitations as micromanipulation because of the overlapping size of Eimeria spp. Chancaerization is further limited by the lack of suitable size/shape standards for flow cytometry-Key words. Cell sorter. Eimeria spp., oocyst shape, oocyst size.     

Variation in Eimeria oocyst count and species composition in weanling beef heifers

Rectal fecal samples were collected daily on 10 consecutive days in November 2004 from 11 weaned beef heifers to assess daily variation in fecal oocyst count and species composition. Subsequent samples were collected from the same animals on 15 April 2005 and 9 June 2005. Oocyst numbers were determined by the modified McMaster's test, and species were identified by examination of oocysts recovered with the Wisconsin sugar flotation technique. Soil samples were collected from the heifer pasture on 8 June 2005, and oocysts were quantified and identified to species. Mean fecal oocyst counts varied little at all sampling dates ranging from 134-377 oocysts/g. Ten Eimeria spp. were identified in fecal samples collected in November and April and 11 in June. Eimeria bovis was the most common species identified at all samplings. Mean species composition showed little variation during the 10-day sampling period in November, remained similar in April, and varied slightly in June. Twelve Eimeria spp. were identified in soil samples in proportions similar to those seen in fecal samples. The results indicate that clinically normal weanling beef heifers are likely to be infected with a diverse, but relatively stable, community of Eimeria spp.     

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.     

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.     

Eimeria acervulina, E. brunetti, and E. maxima: immunity in chickens with low multiple doses of mixed oocysts.

Young chickens inoculated with multiple low doses of mixed oocysts of Eimeria acervulina, E. brunetti, and E. maxima had a high level of resistance to reinfection with a mixed challenge dose on Day 28, Day 84, or Day 140. Immunity was enhanced when the number of immunizing doses was increased from three to four. Resistance was also high in birds maintained on a proprietary mixture of amprolium, ethopabate, and sulphaquinoxaline (Pancoxin-Merck, Sharp and Dohme Ltd.) during immunization, although immunity to E. acervulina was lower in these birds. Oocyst production was lower in birds given mixed infections as compared with that of birds given pure infections with similar doses of oocysts. Competition between species did not inhibit the development of immunity in birds given low doses of mixed oocysts.     

Eimerian life cycles: the patency of eimeria vermiformis, but not Eimeria pragensis, is subject to host (Mus musculus) influence

The ability of Eimeria vermiformis and Eimeria pragensis to produce oocysts in primary infections is influenced by host factors. Oocyst production and the duration of patency were determined for each species in: NIH mice, normal or immunosuppressed (by X-irradiation or injection of cortisone acetate), and strains of mice: nu/+, nu/nu; BALB/c, C57BL/6 known (E. vermiformis), or suspected (E. pragensis), to be of contrasting susceptibility to infection. The effect of induced or genetic susceptibility on the reproduction of E. vermiformis resulted in increases in both oocyst production and the duration of patency. Eimeria pragensis was less affected, with smaller increases in the numbers of oocysts produced and no significant prolongation of patency. The implications of these findings with respect to eimerian life cycles are discussed.     

Cultivation of Eimeria tenella in Japanese quail embryos (Coturnix coturnix japonica).

Complete development of Eimeria tenella in Japanese quail embryos was observed. Sporozoites were inoculated into the allantoic cavity of 7-day-old Japanese quail embryos (Coturnix coturnix japonica), after which the infected embryos were incubated at 41 C. In the chorioallantoic membrane mature first generation schizonts, mature second generation schizonts, and gametes were detected at 48 hr postinoculation of sporozoites (PI), 84 hr PI, and 126 hr PI, respectively. Mature gametes and zygotes were found at 132 hr PI, and oocysts were detected at 138 hr PI. Mortality of embryos increased with increment of inoculum size of sporozoites. LD50 was 1.7 x 10(2) sporozoites. Oocyst production was also dependent on inoculum size. Oocysts harvested from embryos sporulated. The oocysts were inoculated into 13-day-old chickens, and oocysts, capable of sporulating normally, were recovered from ceca 7 days after inoculation.     

Immunity to Eimeria separata (Apicomplexa: Eimeriina): expose-and-challenge studies in rats

Oocyst production of Eimeria separata of the rat was determined in initial and challenge infections. The total number of oocysts produced was no higher at 100 oocysts than at 1,000, 10,000, or 100,000 in initial infections. In 2 experiments, the reproductive index with 100 oocysts was 17,000 and 34,000, respectively. On challenge with 10,000 oocysts, there was 91% protection at 100 oocysts. There was slightly lower protection at 10,000 and 100,000 oocysts, 72% and 81%, respectively, and t-values suggest lower protection at these higher inocula.     

Eimeria filamentifera sp. n. from the Snapping Turtle, Chelydra serpentina (Linné), in Iowa

SYNOPSIS. Oocysts of Eimeria filamentifera sp. n. were isolated from 2 of 2 snapping turtles, Chelydra serpentina (Linné), in Iowa. The ovoid to ellipsoid oocysts of this species averaged 23.2 × 18.6 μm and had a length/width ratio ranging from 1.1 to 1.6. The ellipsoid sporocysts averaged 14.0 × 7.7 μm and had papillose, filament-bearing Stieda bodies. Oocyst and sporocyst residua were present, but micropyle and polar granule were not. This appears to be the 2nd species of Eimeria reported from snapping turtles, with E. chelydrae being the first.     

Eimeria sinaitae n. sp. (Apicomplexa: Eimeriidae) from the rock agama (Agama sinaita) in Saudi Arabia

Eimeria sinaitae n. sp. is described from the gall bladder of Agama sinaita from Wasie, Saudi Arabia. Sporulated oocysts are elongate-ellipsoid 34.4 x 22.0 (29.0-40.0 x 17.4-24.5) micron. Oocyst wall is smooth, greenish yellow, 1.2 (1.0-1.4) micron thick, and two-layered. Micropyle, polar granule, and oocyst residuum are absent. Sporocysts are ellipsoid 11.4 x 7.6 (9.8-15.0 x 6.7-9.0) micron. Sporocyst residuum is present. The sporocysts lack a Stieda body. Sporozoites are crescent-shaped, blunt at one end and tapered at the other. Eimeria species from Agamidae are compared.     

Coccidia of Brazilian mammals: Eimeria corticulata n. sp. (Apicomplexa: Eimeriidae) from the anteater Tamandua tetradactyla (Xenarthra: Myrmecophagidae) and Eimeria zygodontomyis n. sp. from the cane mouse Zygodontomys lasiurus (Rodentia: Cricetidae)

Feces from a specimen of Tamandua tetradactyla (Linn.) from Portel, Pará State, north Brazil, contained two different coccidial oocysts; one identified as Eimeria tamanduae Lainson 1968, and the other as a new species, described here as Eimeria corticulata n. sp. Oocysts of E. corticulata are ellipsoidal, 37.4 x 30.4 (31.2-43.7 x 23.7-35.0) microns, shape index (length/width) 1.2 (1.0-1.5). Oocyst wall 2.5-3.7 microns thick and composed of two layers; an outer thick, brown-yellow one with radial striations, and a thin inner smooth one: no visible micropyle. Oocyst residuum a large globule of about 10.7 x 10.3 microns, usually accompanied by a number of smaller attached globules. Sporocysts ellipsoidal, 21.0 x 11.0 (20.0-22.5 x 10.0-12.5) microns, with a conspicuous Stieda body; shape index 1.9 (1.6-2.2). Sporocyst residuum a small number of scattered granules: sporozoites 18.7 x 5.0 microns, with a large posterior refractile body. Eimeria zygodontomyis n. sp. is described in feces from Zygodontomys lasiurus (Lund) from the Serra dos Carajás, Pará. Oocysts ellipsoidal to cylindrical, 16.5 x 12.0 (13.7-18.7 x 11.2-12.3) microns, shape index 1.4 (1.2-1.5). Wall colorless, smooth, single-layered and about 0.6 micron thick: no micropyle. No oocyst residuum, but a polar granule of about 1.8 x 1.0 microns is sometimes present. Sporocysts ellipsoidal, 8.4 x 5.5 (7.5-8.7 x 5.0-6.2) microns, shape index 1.5 (1.4-1.7), with a thin colorless wall and a delicate Stieda body. Sporozoites enclose a compact residuum of about 2.5 x 3.7 microns.     

Eimeria ochrogasteri n. sp. from the Prairie Vole Microtus ochrogaster*

SYNOPSIS. Eimeria ochrogasteri n. sp. (Coccidia, Eimeriidae) from a prairie vole Microtus ochrogaster (Rodentia, Cricetidae) is described. This is the first recorded coccidium in prairie voles. Sporulated oocysts spherical to ellipsoidal, mean 24.0 by 20.5 μ. Oocyst wall double, outer layer thick, yellow-brown, deeply pitted, inner layer clear. Oocyst residuum varies from many small globules to a coalesced group of large and/or small globules. Polar granule present. Micropyle absent. Sporocysts ovoid with “capped” Stieda body, mean 12.3 by 8.2 μ. Sporocyst residuum present. Sporozoites average 14.9 by 2.9 μ with spherical anterior and oblong posterior refractile globules. This species was found in 1 of 71 voles from Weld county, Colorado.     

Eimeria lancasterensis Joseph, 1969 and E. confusa Joseph, 1969 from the Grey Squirrel Sciurus carolinensis*

SYNOPSIS. Forty grey squirrels Sciurus carolinensis were examined for coccidia during a 2-year period. Eimeria lancasterensis was found in all of them. The ellipsoidal oocysts of this species averaged 24.6 by 14.6 μ. They had no micropyle or oocyst residuum. A polar body was present. The sporocysts averaged 14.1 by 8.4 μ. The endogenous phases of the parasite were found in the epithelial cells of the villi thru the entire length of the small intestine. E. confusa was found in one of 40 squirrels. The oocysts of this species were subspherical, occasionally ellipsoidal or rarely spherical; they averaged 33.2 by 26.7 μ. Oocyst residuum and micropyle were absent. Polar granules ranged in number from 0–5. The sporocysts averaged 19.6 by 12.1 μ. The prepatent period for this species was 7–8 days and the patent period 6–13 days. E. ontarioensis was found in 3 of the 40 squirrels.     

Eimeria sinaitae n. sp. (Apicomplexa: Eimeriidae) from the Rock Agama (Agama sinaita) in Saudi Arabia

Eimeria sinaitae n. sp. is described from the gall bladder of Agama sinaita from Wasie, Saudi Arabia. Sporulated oocysts are elongate-ellipsoid 34.4 times 22.0 (29.0–40.0 times 17.4–24.5) μm. Oocyst wall is smooth, greenish yellow, 1.2 (1.0–1.4) μm thick, and two-layered. Micropyle, polar granule, and oocyst residuum are absent. Sporocysts are ellipsoid 11.4 times 7.6 (9.8–15.0 times 6.7–9.0) μm. Sporocyst residuum is present. The sporocysts lack a Stieda body. Sporozoites are crescent-shaped, blunt at one end and tapered at the other. Eimeria species from Agamidae are compared.     

Eimeria juniataensis sp. n. (Protozoa: Eimeriidae) from the map turtle, Graptemys Geographica, in Pennsylvania.

Eimeria juniataensis sp. n. is described from the map turtle, Graptemys geographica, in Pennsylvania. The spherical to subspherical oocysts of E. juniataensis are 11.5 to 18.5 mu by 11.5 to 16.5 mu (mean, 13.5 by 12.9mu). The broadly fusiform sporocysts are 7.5 to 10 mu by 4 to 6.5 mu (mean, 8.3 by 5.0mu) and a Stieda body is present. Oocyst and sporocyst residua are present. This is the first report of an eimerian species from Graptemys.     

Coccidia from the Tiger Salamander, Ambystoma tigrinum, in Northeastern Colorado and in Northern New Mexico

SYNOPSIS. Oocysts of Eimeria ambystomae Saxe, 1955, Eimeria microcapi sp. n., and Eimeria urodela sp. n. are described from the tiger salamander, Ambystoma tigrinum , collected in Colorado and New Mexico. The oocysts of E. ambystomae are ellipsoid, 29.8 × 17.3 (24–38 × 15–25) μm, and the sporocysts lanceolate, 22.6 × 5.4 (16–27 × 5–7) μm. Oocyst and sporocyst residua are present, but not a polar granule and a micropyle. The oocysts and sporocysts of E. microcapi are ellipsoid, measuring respectively 38.1 × 25.3 (35-41 × 23-26) μm and 18.1 × 7.4 (16-19 × 6–8) μm. Oocyst and sporocyst residua, a micropyle (mean 3 μm), and a distinct micropyle cap (2 μm high) are present, but not a polar granule. The oocysts of E. urodela are spheroid, 22.2 (14-26) μm, and the sporocysts lanceolate, 16.3 × 5.8 (12-19 × 4-7) μm. Oocyst and sporocyst residua are present, but a polar granule and a micropyle are absent.     

Polymorphism of eimerian oocysts can be a problem in naturally infected hosts: an example from subterranean rodents in Bolivia

Since 1986, 364 tuco-tucos (Ctenomys spp.) representing 7 species were collected from 16 major collecting areas representing at least 4 distinct ecological habitats in Bolivia, South America. All were examined for coccidia, and 125 (34%) had oocysts in their feces including 84 of 236 (36%) Ctenomys boliviensis from tropical palm/savanna habitats; 1 of 3 (33%) Ctenomys conoveri from a chaco thorn forest; 3 of 7 (33%) Ctenomys frater from medium altitude grass habitats; and 6 of 8 (75%) Ctenomys lewisi and 31 of 35 (88%) Ctenomys opimus from high altitude/puna habitats. None of 3 Ctenomys leucodon (high altitude/puna) or 72 Ctenomys steinbachi (tropical palm/savanna) were passing oocysts when examined. The 5 infected host species all had oocysts of Eimeria opimi Lambert, Gardner, and Duszynski, 1988, in their feces. These oocysts and their sporocysts varied greatly in size, both within and between host species, but qualitative characters (e.g., residua and wall texture) remained constant. Our conclusion, that all oocysts seen were E. opimi, was supported by multigroup discriminant analysis of 256 individual oocysts, 30-67 selected randomly from each Ctenomys sp. Minimum polygons enclosing the centroid (= multivariate mean) and the spread of individuals for each species group (OTU) showed significant overlap in discriminant space, and Geisser classification showed a 55% miss rate of individuals being classified into the wrong OTUs. Thus, oocyst and sporocyst lengths and widths cannot be used to separate morphotypes of E. opimi from different Ctenomys spp. from different geographic regions of Bolivia.     

Structure and Respiration of Sporulating Eimeria stiedae and E. tenella Oocysts*

SYNOPSIS. The structure and respiration of sporulating Eimeria tenella and E. stiedae oocysts was compared. Oocysts were purified by Clorox digestion and incubated at 29 C. The sporulation events were similar for both species, except for the spindle stage, altho sporulation took longer in E. stiedae. Sporulating oocysts go thru the process with some degree of synchrony. Oocyst respiration was measured polarographically. An early increase in respiratory rate in both species was followed by a depression in rate that correlated with the appearance of the early spindle stage. The rate again increased and then decreased toward a base rate during and after completion of sporulation. E. tenella oocysts incubated in air remained viable 4 times longer than those incubated in nitrogen. E. tenella sporozoites excysted and remained motile in the presence of sodium cyanide. These facts suggest that E. tenella sporozoites are facultative anaerobes.     

Description of a new Eimeria sp. and associated lesions in the kidneys of double-crested cormorants (Phalocrocorax auritus)

A new Eimeria sp. is described as the cause of an outbreak of renal coccidiosis in double-crested cormorants (Phalacrocorax auritus) in Georgia. Sporulated oocysts were spherical to subspherical and measured 16.1 x 16.5 (13.8-18 x 14-19) microm, with an average length-width ratio of 1:1. Oocyst wall was thin (1 microm), greenish, and pitted on the outer surface. Micropyle, micropylar cap, Stieda body, and polar bodies were absent. Small oocyst residuum (4-8 granules) was usually absent but occasionally present. Sporocysts were oval and measured 6.6 x 9.3 (6-7 x 8-10.5) microm, with an average length-width ratio of 1:1.4. A sporocyst residuum was present, located in between sporozoites, and was composed of numerous granules of unequal size. A small refractile body was present in each sporozoite. Collecting duct and distal renal tubular epithelial cells were distended by large oocysts in their cytoplasm, and many oocysts were present in the lumen of dilated tubules. Various stages of meronts, gamonts, and developing oocysts were present in other renal tubular epithelial cells. Multiple infections of parasitized cells were frequently observed, with cells containing up to 12 gamonts or developing oocysts. The importance of this Eimeria sp. on double-crested cormorant populations is not known. But in this case, significant lesions and mortality were associated with infection.