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. mitis caused difficulty in separation from the remaining fine debris. This technique was useful for studying the mixed isolates by bit-map gating had the same limitations as micromanipulation because of the overlapping size of Eimeria spp. Characterization is further limited by the lack of suitable size/shape standards for flow cytometry.     

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.     

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.     

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.     

Oocyst production of Eimeria lettyae in northern bobwhites following low-dose inoculations

Inoculation of northern bobwhite quail ( Colinus virginianus ) with low doses of Eimeria lettyae oocysts stimulates a protective immune response, suggesting immunization may be an option for controlling coccidiosis. However, the oocyst production of inoculated birds could be considerable, leading to subsequent outbreaks. To determine the oocyst production following inoculation with E. lettyae, we orally infected 12-wk-old bobwhites with 100, 1,000, or 10,000 sporulated oocysts. Fecal materials were collected on days 5-9 post-inoculation, and total oocyst production was counted in McMaster chambers. Oocyst production/bird was 49.75, 89.5, and 436 × 10(6) for 100, 1,000, or 10,000 oocysts administered, respectively. Estimated oocysts produced/oocyst administered was 49.75, 8.95, and 4.36 × 10(4) for 100, 1,000, or 10,000 oocysts administered, respectively. These findings not only illustrate the crowding effect of larger oocyst inocula but also illustrate the fecundity of E. lettyae at low doses. This suggests that successful immunization of bobwhites against coccidiosis with live vaccines might require attenuated strains with reduced reproductive potential.     

Eimeria (Protozoa: Eimeriidae) of the Cottontail Rabbit Sylvilagus audubonii in Northeastern Colorado, with Descriptions of Three New Species

SYNOPSIS. All of 100 cottontail rabbits Sylvilagus audubonii were found to be infected with 1-6 species of Eimeria. Three new species, E. audubonii, E. neoirresidua and E. poudrei are described from this host. Sub-spherical oocysts of E. audubonii average 21.2 by 17.1 μ; polar body, micropyle, oocyst residuum and sporocyst residuum are all absent; ellipsoidal sporocysts average 12.9 by 5.8 μ. Ovoid to ellipsoidal oocysts of E. neoirresidua average 25.7 by 17.9 μ; polar body and oocyst residuum are absent; micropyle and sporocyst residuum are present; ellipsoidal sporocysts average 14.5 by 6.4 μ. Ovoid to ellipsoidal oocysts of E. poudrei average 26.0 by 18.1 μ; polar body is lacking; micropyle, oocyst residuum and sporocyst residuum are present; ellipsoidal sporocysts average 14.4 by 6.4 μ. Three species of Eimeria previously described in the literature, E. maior Honess, 1939, E. media form honessi Carvalho, 1943 and E. environ Honess, 1939 are redescribed. A detailed structural and statistical analysis of each species is presented with at least 200 sporulated oocysts measured in each instance. A host list and a key to the Eimeria of cottontails is given. The use of detailed studies of oocyst size and structure as a tool for specific diagnosis of the Eimeria of cottontails is discussed.     

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.     

Infections with Eimeria maxima and Eimeria acervulina in the fowl: effect of previous infection with the heterologous organism on oocyst production.

Judged by oocyst production, infections with Eimeria acervulina in birds immunized with E. maxima were consistently higher than in birds which had not been immunized. Oocyst production of E. maxima in birds previously infected with E. acervulina was greater, in three out of four experiments, than in control chickens, but some of the differences were slight. The findings are discussed but no satisfactory explanation can be offered. In general there was little or no difference between the oocyst production of the individual species when present as single or mixed infections.     

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.     

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 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.     

Prevalence of Eimeria macusaniensis (Apicomplexa: Eimeriidae) in midwestern Lama spp

To compare the prevalence of Eimeria macusaniensis among midwestern llamas (Lama glama), alpacas (Lama pacos), and guanacos (Lama guanicoe), feces were obtained from Lama spp. in 10 states between October 1989 and February 1996. Feces were examined by centrifugal flotation in sugar solution (specific gravity--1.28-1.30), and oocysts were quantified by a modified McMaster method. Data were compared by host species and age classifications. Typical oocysts occurred in samples from 28% of 76 herds and 10.4% of 443 animals including 12% of 301 llamas, 7% of 115 alpacas, and 7.4% of 27 guanacos. Prevalence was significantly greater (P = 0.009) in animals < 1 yr of age in comparison to older animals for llams (22.1 v.s. 8.5%) and for all Lama spp. combined (17.1 vs. 8.4%). Fecal oocyst abundance was significantly greater (P = 0.001) in llamas < 1 yr of age in comparison to older llamas (30 vs. 16 oocysts per g of feces). Fecal oocyst intensities did not differ significantly. Prevalence in both age groups of midwestern llamas was greater than previously reported for llamas in the western United States. Prevalence in midwestern alpacas < 1 yr of age was lower than reported for alpacas of similar age in South America, but oocyst intensities were similar. These results indicate that infection with E. macusaniensis is more common in Lama spp. in North America than previously recognized.     

Quantification of the crowding effect during infections with the seven Eimeria species of the domesticated fowl: its importance for experimental designs and the production of oocyst stocks.

The 'crowding effect' in avian coccidia, following administration of graded numbers of sporulated oocysts to na?ve hosts, is recognisable by two characteristics. First, increasing doses of oocysts give rise to progressively higher oocyst yields, until a level of infection is reached (the 'maximally producing dose') above which further dose increases result in progressive decreases in oocyst yields. Second, the number of oocysts produced per oocyst administered (the 'reproductive potential') tends to decrease as the oocyst dose is increased. The dose that gives the maximal reproductive potential is the 'crowding threshold' and doses exceeding this are 'crowded doses'. Graded doses of Eimeria acervulina, Eimeria brunetti, Eimeria maxima, Eimeria mitis, Eimeria necatrix, Eimeria praecox or Eimeria tenella were given to chickens of the same breed, sex and age, reared on the same diet, under identical management. The two characteristics of the crowding effect were demonstrated graphically and, by interpolation, the estimated crowding thresholds were 903, < or =16, 39, < or =14, < or =16, < or =16 or 72 sporulated oocysts, respectively, for the seven Eimeria species enumerated above. This is apparently the first report of definitive experiments to quantify a crowding effect in E. brunetti, E. maxima, E. mitis, E. necatrix and E. praecox. Maximum experimental reproductive potentials were considerably lower than the theoretical reproductive potentials for all seven species. The interaction between availability of host intestinal cells and immunity contributing to the crowding effect is discussed. Standard curves obtained under specified conditions should be used to estimate appropriate infective doses for experimental designs or in vivo production of oocyst stocks. For experiments on effects of chemotherapy or immunisation on oocyst production, an infective dose lower than the crowding threshold should be used. For efficient production of laboratory or factory oocyst stocks, the maximally producing dose (which is greater than the crowding threshold), should be used.     

Eimerians in Harvest Mice, Reithrodontomys spp., from Mexico, California and New Mexico, and Phenotypic Plasticity in Oocysts of Eimeria arizonensis

ABSTRACT Between May 1979 and August 1991, 48.7% (57/117) of the harvest mice ( Reithrodontomys spp.) examined from 10 localities in Mexico, California and New Mexico had coccidian oocysts in their feces. A total of 46.7% (49/105) of the Reithrodontomys megalotis examined were positive for coccidian oocysts; this included samples from five states in Mexico (47.1%, 8/17), three counties in California (66.7%, 4/6) and two counties in New Mexico (45.1%, 37/82); 66.7% (8/12) of the Reithrodontomys montanus from one county in New Mexico also were infected. Only two coccidian species, Eimeria arizonensis and Eimeria langebarteli , were found in these hosts. Oocysts of E. langebarteli were found only in R. megalotis : in all three infected mice from Madera County, California, in the only mouse from San Bernardino County, California, and in 63% (5/8) of the infected mice from four states in Mexico. Oocysts of E. arizonensis were found in R. megalotis in Mexico, California, and New Mexico and in R. montanus from New Mexico. Sporulated oocysts of E. langebarteli differed slightly from those in previously published reports by having wider oocysts and larger sporocysts. Sporulated oocysts of E. arizonensis were variable in size, with those recovered from R. montanus significantly larger in length and width and sporocyst width than those from R. megalotis . The structure of the oocyst residuum was polymorphic, both within and between host species, and within the same mouse; it could appear as one large globule, two globules, several to many smaller globules, or as a compact mass of many small granules. Oocysts with a variable residuum were larger than those with one globule in all oocyst/sporocyst dimensions. Only 9% (5/57) of the infected mice were discharging oocysts of both eimerians when examined.     

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.     

Effects of time and watershed characteristics on the concentration of Cryptosporidium oocysts in river water.

Water samples were collected from four locations on two rivers in Washington State and analyzed by membrane filtration-immunofluorescence assay to establish Cryptosporidium oocyst concentrations. Sampling locations were selected to evaluate effects of watershed character, from pristine mountain to downstream agricultural, on oocyst concentrations. Samples were collected at six biweekly intervals from late June to early September, with two additional sets of five samples taken on separate days (one set taken at bihourly intervals and one set taken simultaneously). Cryptosporidium oocysts were found in 34 of 35 samples at concentrations ranging from about 0.2 to 65 oocysts per liter. Oocyst concentrations were highest early in the sampling period, when they were influenced by postrainfall runoff, and decreased through the summer months. Oocyst concentrations found in ten samples collected on two days (5 samples per day) showed no short-term variations. Oocyst concentrations and oocyst production per square mile (ca. 2.6 km2) of watershed found in water draining a controlled public water supply watershed were the lowest observed. The concentrations and production rates for drainage from an adjacent, comparable, but uncontrolled watershed were nearly 10 times higher. The concentration and production rates of the downstream area influenced by dairy farming were nearly 10 times higher than rates at the upstream stations. The data showed clearly that oocyst concentrations were consistently observed above the detection limit of the analytical method, about 0.1 oocysts per liter; that oocyst concentrations were continuous as opposed to intermittent; and that watershed character and management affected surface water oocyst concentrations significantly.