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An isolate of Eimeria meleagridis Tyzzer, 1927 was obtained by harvesting oocysts from the ceca of a turkey from northwest Arkansas and a pure line was established by infecting birds with a single oocyst. Oocysts were first produced in the ceca of infected birds from 102 to 108 hr after inoculation and were of similar size (mean length × width, 24.9 × 17.0 μm) to those of Eimeria adenoeides Moore and Brown, 1951 and Eimeria gallopavonis Hawkins, 1952. The line was identified as E. meleagridis based upon the development of large schizonts in the midintestine, and small schizonts in the ceca. Two generations of large schizonts were found 48 and 72 hr after infection, and at least two generations of small schizonts were found from 60 to 108 hr after infection. An inoculum of 2 × 105 oocysts was found to cause a significant reduction in weight gain from days 0–3 and 0–6 after infection, suggesting that the significance of this species of Eimeria as a pathogen of turkeys should be reassessed.

The Guelph strain of Eimeria adenoeides was obtained from a commercial turkey flock in Ontario, Canada, in 1985. Single oocyst derived lines of E. adenoeides were propagated, and one of them used to re-describe biological and morphological features of E. adenoeides in the turkey. Oocysts of this strain are within the lower size ranges in the original species description reported by Moore and Brown (1951); oocysts of the Guelph strain averaged 18.7 ± 1.4 μm (16.7-22.5) by 14.3 ± 0.9 μm (13-16.2, n = 30) with a shape index (SI) of 1.3 ± 0.1. It is possible that the original species description was based, at least in part, on a mixed culture of two or more Eimeria species. Immature first-generation meronts of E. adenoeides Guelph strain were observed histologically at 32 h post-infection in the ileum and cecal neck. Early studies reported only two asexual generations suggested that first asexual cycle observed at 32 h post-infection was overlooked. In the present study, three asexual generations were observed before the start of gametogony. The Guelph strain is also characterized by a prepatent period of 112 h. The Guelph strain of E. adenoeides is a highly pathogenic coccidium that forms classic cecal lesions, including prominent caseous cecal cores, during moderate to severe infections. The maximum output of oocysts (1.77 × 10(7) per bird) was obtained from birds inoculated with 1 × 10(3) oocysts; maximum fecundity (1.55 × 10(5) oocyst shed per oocyst inoculated) was obtained with an inoculation of 1 × 10(2) oocysts, but fecundity dropped dramatically as the inoculation dose increased. To promote stability of the E. adenoeides species concept, neotype specimens (a parahapantotype slides series and phototype) have been designated and deposited for future reference.

The progress and transmission of blackhead disease in chickens was studied in battery cages and floor pens in the absence of vectors. Two-week-old chicks were inoculated intracloacally with Histomonas meleagridis and allowed to commingle with others in floor pens. There was no confirmed transmission of blackhead to other birds in the pen, whether stocked at 10% or 25% with infected birds. A second experiment evaluated the effects of feed restriction of chickens on spread of blackhead within floor pens. Inoculated seeder birds had severe cecal lesions of blackhead at necropsy, regardless of feed restriction. Uninoculated birds did not develop lesions by the time of necropsy at 42 days of age, regardless of whether full-fed or limited by skip-day feeding. Chickens inoculated intracloacally with H. meleagridis and placed in battery cages became infected and had cecal lesions of blackhead, but few liver lesions. Chickens allowed to commingle with the inoculated birds in batteries had no lesions of histomoniasis at necropsy 2 wk postinoculation. Coccidial oocysts from turkeys (Eimeria adenoeides) were inoculated along with H. meleagridis from cultures to test the effects of sporozoite penetration in the ceca on progress of blackhead disease. Histomoniasis was not worsened by the interaction with sporozoites, as shown by unchanged severity of cecal lesions, the number of birds showing liver lesions, or the overall number of positive birds. Overall, blackhead infections showed no inclination to spread from bird to bird under conditions of these studies, in contrast to what has been reported for turkeys. These results suggest that the dynamics of blackhead transmission in chickens differs significantly from that of turkeys, where transmission from bird to bird is rapid and effective in the absence of vectors.

Coccidiosis is one of the most prevalent diseases in poultry. The objective of the current study was to determine the effects of floor heating when poults were artificially infected with Eimeria regarding the outcome of the infection and secondary effects on litter quality and health of the foot pad. Two trials were performed. In each trial, 4 groups of 2-wk-old turkeys were reared for a 4-wk period. All birds were fed ad libitum identical pelleted diets without any anticoccidial additive. The first 2 groups were housed on dry wood shavings, with and without floor heating; the other 2 groups were housed on wet wood shavings (35% moisture, achieved by adding water as required), with and without floor heating. Two birds only (primary seeder birds; the other 18 birds were nominated as secondary infected birds) in each of the 4 groups were experimentally infected orally with Eimeria adenoeides (˜50,000 oocysts/bird). The number of oocysts eliminated via excreta was determined repeatedly. On d 42, each bird not only was scored macroscopically for coccidial lesions in the cecum but also oocyst counts were determined in the cecal contents. Finally, the foot pads were assessed weekly for external scoring and on d 42 of life for histopathological scoring. Although the counts of oocysts in seeder birds were almost identical (d 6 postinoculation), oocyst counts in excreta of secondary birds were markedly reduced in both trials when the poults were not exposed to wet litter. Moreover, in both trials using floor heating with exposure to wet litter resulted in a higher oocyst count in the excreta of secondary infected birds (3.72/3.92 in trials 1 and 2) on d 24 postinoculation compared with the other groups. Using floor heating resulted in significantly decreased foot pad dermatitis scores compared with groups housed without floor heating. In conclusion, the differences in oocyst counts indicate that the process of sporulation is affected by both the moisture and the temperature of the litter.

Cells were dispersed from cecal tissues of 1- to 2-day-old turkeys using a mixture of collagenase and dispase to enrich for epithelial cells. The initial culture produced from these cells (TCC) appeared to be heterogeneous, but, as the cells were cultured through 25 passages, they assumed a more fibroblastic appearance. Cellular invasion of the TCC by two species of turkey coccidia, Eimeria adenoeides and Eimeria meleagrimitis, was not enhanced, as compared with invasion in turkey kidney cells (TKC), the cell culture system standardly used to study the avian coccidia in vitro. However, early development by one of the species, E. meleagrimitis, was markedly increased in the TCC (Passages 6 through 19) over that in TKC. Thirty to 42% of the parasites that invaded the TCC developed beyond the sporozoite stage, as compared with 5% development in TKC. Mature first-generation schizonts were observed within 24 hr postinoculation in the TCC, but not until 48 hr in the kidney cells. There was no evidence that development of the second generation was initiated in either the TCC or kidney cells.

The progression of coccidiosis provoked by E. adenoeides was followed in turkey poults given ochratoxin A (OTA) in feed. More heavy and rapid progress of coccidiosis occurred in OTA-treated turkey poults than in those fed an OTA-free diet, as can be seen from the higher levels of oocyst and lesion indices and more rapid mortality in such birds. The concentration of total protein in serum was significantly increased in turkey poults infected with E. adenoeides and simultaneously given OTA. The serum concentration of uric acid was significantly increased in all experimental groups, especially in those exposed to OTA, and most notably in the group additionally infected with E. adenoeides. OTA induced degenerative changes in kidneys, liver and heart as well as depletion of lymphoid tissue in the lymphoid organs and a decrease in body mass. Coccidiosis also induced a growth depression in addition to caecal haemorrhages. Impairment of kidney function, histopathological changes and general growth depression were stronger when turkey poults infected with E. adenoeides were also given OTA., Istraživan je tijek kokcidioze uzrokovan protozoonom E. adenoeides u purića koji su dobivali ohratoksin u hrani. Znatno teži oblik bolesti uočen je u purića pokusne u odnosu na kontrolnu skupinu. Razlika je uočena na razini broja oocista, stupnja oštećenja i bržeg uginuća. Koncentracija ukupnih proteina u serumu bila je također značajno povišena u pokusne skupine. Koncentracija mokraćne kiseline u serumu bila je osobito povišena uskupini invadiranoj kokcidijama i hranjenoj hranom s primiješanim ohratoksinom. Ohratoksin je uzrokovao degenerativne promjene u bubrezima, jetrima i srcu kao i depleciju limfoidnog tkiva te smanjenje tjelesne težine. Kokcidioza je također uzrokovala smanjeni rast, ali i krvarenje u slijepim crijevima. Patohistološke promjene, smanjena funkcija bubrega kao i opće smanjen prirast bili su izraženiji u invadirane skupine koja je u hrani dobivala ohratoksin.

An effective method for the enrichment of two cecal species of the avian Eimeria in mixed cultures has been developed. The method involves the transfer of sporozoites of Eimeria tenella and Eimeria adenoeides from their specific site of invasion in a foreign host bird to the natural host, and markedly increases the numbers of the cecal species in relation to the other species in the mixture. Donor turkeys and chickens were inoculated with mixed cultures containing equal numbers of oocysts of the chicken coccidia, E. tenella + Eimeria acervulina, or the turkey coccidia, E. adenoeides + Eimeria meleagrimitis, respectively. After 8 to 24 h, the cecal tissues of the donor birds were scraped into phosphate-buffered saline and transferred, per os, to the natural host. On Day 6 to 7 posttransfer, numerous oocysts of E. tenella and E. adenoeides were found in the ceca of the recipient chickens and turkeys, respectively. In contrast, only occasional oocysts were found in a few birds in other areas of the intestine.

Plasma epinephrine, norepinephrine, and glucose levels were measured in 3- to 4-wk-old turkey poults that had been infected with the coccidium Eimeria adenoeides. When measured at 6 days postinoculation, the acute stage of the infection, the epinephrine and norepinephrine levels of the infected poults showed a dose-dependent increase over levels in controls, with poults that were most heavily infected having the highest levels. The catecholamines appeared to show a classic stress-elicited response: this response increases with time to high levels during the acute stage of the infection, and then returns to levels approximately the same as those of the controls as the poults recover from the infection. Anorexia, or feed restriction, appeared to be primarily responsible for the increase in norepinephrine. However, epinephrine levels in the infected poults were significantly higher than in their pair-fed, uninfected counterparts, indicating that factors other than reduced feed consumption contributed to the increase. The data indicate that plasma catecholamines are increased during coccidial infection. As pressor compounds, they may contribute to the previously observed changes in blood pressure and heart function in infected poults.

Three-week-old turkey poults receiving 250 μg/kg of dietary aflatoxin displayed prolonged prothrombin times due to decreased fibrinogen levels and depressed activity of factor II. Decreased body weights and increased mortality were also evident. Poults experiencing an Eimeria adenoeides infection had slightly increased prothrombin times that were not corrected by the addition of specific clotting factors. These birds exhibited significant reductions in total plasma protein levels. The combination of E. adenoeides and aflatoxicosis resulted in a significant depression in body weight, altered prothrombin time, and decreased levels of plasma calcium, protein, and fibrinogen. Increased mortality was also observed. E. adenoeides did not affect the hemostatic system of the turkey as severely as does aflatoxin. Turkeys were shown to be highly susceptible to aflatoxin when compared to chickens.

Halofuginone was tested at 0, 1.5, 3.0, or 6.0 ppm in the feed against single and mixed infections of recent field isolates of Eimeria adenoeides, E. meleagrimitis, and E. gallopavonis. Severe infections were produced in unmedicated, infected controls. Weight gains were significantly improved by 1.5 or 3 ppm of halofuginone, and intestinal lesion scores and fecal droppings scores were greatly reduced. Oocyst shedding was reduced 91 to 100% by halofuginone treatments. Treatment with 6 ppm of halofuginone gave good protection but appeared to depress weight gains slightly. Treatment with 3 ppm was optimal for the control of all species of coccidia and for optimum performance.

TO DATE six species of coccidia affecting the turkey have been described. They are Eimeria meleagridis, Eimeria meleagrimitis, and Eimeria dispersa described by Tyzzer (1929), Eimeria gallopavonis described by Hawkins (1950), and Eimeria adenoeides and Eimeria innocua isolated and identified by Moore and Brown (1951 Moore and Brown (1952). It is the purpose of this paper to report upon another new species of turkey coccidia. The exact origin of this species is not known since it was obtained from a mixed culture collected by Dr. Philip Hawkins possibly prior to 1950. When Dr. Hawkins discontinued his studies with turkey coccidia his entire collection was turned over to the authors. In one culture labeled E. gallopavonis was found a subspherical type which contained no refractile body. Subsequent investigation of this type definitely indicated that it represented a new species for which the name of Eimeria subrotunda is proposed. A complete description has already been …

The life cycle and pathogenicity of a strain of Eimeria isolated in Great Britain from turkey poults by single cell inoculation are described and, using the criteria laid down by Tyzzer, the species is identified as E. adenoeides.The life cycle is of the same general pattern as in other Eimeria species, consisting of two asexual and one sexual generations.The organism is highly pathogenic for young poults, a dose of 200,000 oocysts producing 100 % mortality in 3-week-old birds and smaller doses causing reduced weight gain. Birds 11 weeks old resisted a dose of 3 million oocysts.The gross and microscopic pathology of the infection is described. No changes were found in the blood picture.

Sulphaquinoxaline fed at 0·0125%, w/w, in the food principally affects the development of the second generation schizonts of Eimeria adenoeides in turkey poults. Amprolium, at the same concentration, acts on the first generation. Both drugs appear to exert some effect on gametogony. The activity of sulphaquinoxaline against this species is inhibited by p-aminobenzoic acid, and the activity of amprolium by thiamine.We are grateful to Mrs B. M. Mitchell and Miss C. Hitchcock for technical assistance and to Mr A. D. Speed for the histological preparations.

A comparison has been made between the use of clopidol-methyl benzoquate and amprolium-ethopabate mixtures for the prophylactic control of Eimeria adenoeides and Eimeria meleagrimitis in turkeys. Twenty four poults were randomly allocated to each of the following treatments: non contaminated non treated; contaminated non treated; contaminated treated with 100 ppm + 8.35 ppm clopidol methyl benzoquate; contaminated treated with 200 ppm + 16.70 ppm clopidol methyl benzoquate; contaminated treated with 125 ppm + 8 ppm amprolium ethopabate. The amprolium-ethopabate mixture only partially controlled an infection with 50,000 sporulated oocysts of E. adenoeides and 200,000 oocysts of E. meleagrimitis. The clopidol-methyl benzoquate mixture completely controlled such an infection at the higher concentration (200 ppm + 16.70 ppm) and almost completely controlled it at the lower concentration (100 ppm+ 8.35 ppm). Drug withdrawal studies showed that the clopidol-methyl benzoquate mixture is coccidiostatic for the two species studied.

Electrophoretic separation of serum proteins of Eimeria-infected turkeys showed five major fractions with significant decreases in albumin levels and significant increases in the alpha 2- and gamma-globulins as compared with controls. The alpha 1- and beta-globulin levels were similar to those of the controls. Significant differences between infected turkeys and uninfected pair-fed controls suggested that protein changes were not solely due to the anorexia associated with Eimeria infections. The increase in alpha 2-globulins was not due to an increase in ceruloplasmin. Indirect fluorescent antibody tests using serum from infected poults as an antibody source were positive, indicating that at least part of the increase in the gamma-globulin fraction was due to the production of immunoglobulins. Electrophoretic separation of plasma lipoproteins showed three to four major bands with a sharp decrease in the portomicrons among infected poults. There was no increase in the pre-beta-lipoproteins (which have the same electrophoretic mobility as the alpha 2-globulins), indicating that the increase in alpha 2-globulins was not due to an increase in the lipoprotein part of this fraction.

Monensin at 60, 80 or 100 ppm provided sufficient early protection against coccidiosis in floor-pens seeded with a mixture of Eimeria adenoeides, E. gallopavonis and E. meleagrimitis oocysts to prevent mortality due to coccidiosis and permit satisfactory weight gains. Unmedicated poults suffered 7% mortality and significantly (P

Floor-pen studies were conducted in three geographic locations to study the efficacy of halofuginone (3.0 ppm in feed) against important species of coccidia in turkeys. Severe coccidiosis exposure was obtained in studies in Georgia and Colorado and mild coccidiosis in Wisconsin. Coccidiosis caused by contamination of the feed with oocysts of Eimeria adenoeides, E. meleagrimitis, and E. gallopavonis was almost completely controlled by halofuginone at 3 ppm, even though intestinal lesion scores and weight loss were high in two studies. Halofuginone was also highly effective in preventing buildup of infection in pens contaminated by indirect means (movement by caretakers through the pens, etc.). Weight gains and feed conversion were best in poults receiving halofuginone, whether directly or indirectly exposed. There was no evidence that halofuginone caused any untoward reactions in the poults, even though the drug was fed until turkeys reached market weight.

The pathogenic effects of infection with freshly sporulated oocysts of E. adenoeides and E. meleagrimitis were studied in 3-week-old turkey poults. The high pathogenicity of E. adenoeides and E. meleagrimitis was confirmed. Small doses of oocysts led to depressed weight gains and a high production of oocysts. The severity of infection increased progressively as the dose increased. Mortality tended to coincide with schizogony at high levels of infection and with gametogony at low levels of infection. The acute phase of the disease lasted from the third or fourth day to the sixth or seventh day after infection. The pathogenic effect of E. adenoeides appeared greater than that of E. meleagrimitis in birds fed an equivalent number of oocysts. Infection with E. adenoeides is recognised by haemorrhagic enteritis in the distal part of the small intestine, the large intestine and the proximal part of the caeca; lesions caused by E. meleagrimitis are confined to the proximal part of the small intestine.

An outbreak of coccidiosis with high mortality is reported in 30-day-old commercial turkeys. Grossly, a severe typhlitis with a large fibrino-necrotic core was present. Large numbers of oocysts were observed in caecal smears. The location and the severity of the lesions and the oocyst morphology were strongly suggestive of Eimeria adenoeides infection. This species has already been reported in turkey flocks in Italy, but it is rarely responsible for clinical coccidiosis and severe lesions with high mortality. Other caecal parasitic infections are considered in differential diagnosis.

Experimental Infection of Turkey Poults with Eimeria Adenoeides (Moore and Brown, 1951) Isolated from a Natural Case in Great Britain

SUMMARY Changes in levels of plasma xanthophyll, plasma retinol, liver retinol, and liver retinylpalmitate in poults infected with Eimeria meleagrimitis or E. adenoeides were measured by high-performance liquid chromatography. Infection with either species significantly reduced total plasma carotenoids; the reduction was due largely to a decrease in xanthophyll to approximately 50% of control levels on the day of maximum effect. In addition, E. meleagrimitis infection decreased plasma retinol levels from 1.84 ,g/ml plasma (controls) to 1.00 ,ug/ml plasma on day 6 postinoculation. Concomitantly, liver retinol levels fell from 0.23 ,ig/mg protein (controls) to 0.15 jg/mg protein, and liver retinylpalmitate levels fell from 0.74 /ug/mg protein (controls) to 0.44 jg/mg protein. In contrast, Eimeria adenoeides infection caused little change in either plasma or liver retinol levels.

SUMMARY Physiologic characteristics were measured on day 7 postinoculation (PI) in 2-week-old turkeys inoculated with 10 Eimeria adenoeides oocysts, in pair-fed controls, and in control turkeys fed ad libitum. Pathophysiologic responses were measured in turkeys inoculated with 7 X 104 LE. adenoeides oocysts or 1.2 X 105 E. meleagrimitis oocysts and necropsied 2, 4, 6, 8, 10, 15, and 21 days PI. At day 7 PI, weight gains and heart weights (as a percentage of body weight) of E. adenoeides-infected turkeys were significantly lower and plasma glucose levels significantly higher than those of pair-fed counterparts. Plasma carotenoid and protein levels of the infected turkeys were significantly lower than those of the controls fed ad libitum and were consistently, but not always significantly, lower than those of the pair-fed controls. Significant responses were first observed in E. meleagrimitis-infected turkeys on day 4 PI and in E. adenoeides-infected birds on day 6 PI. Birds infected with both species commonly had reduced weight gains, heart weights, and plasma carotenoid levels and increased plasma aspartate aminotransferase levels. Plasma carotenoids in E. adenoeides-infected turkeys were significantly reduced on day 6 P1 only, whereas plasma carotenoids in E. meleagrimitis-infected turkeys were sometimes reduced significantly as early as day 2 PI and remained significantly lower than control values through day 21 PI.

SUMMARY The osmotic fragility of red blood cells (RBC) from Eimeria adenoeides-infected turkey poults was cor ipared with that of RBC from control and water-deprived poults. At different hypotonic NaCI concentrations, lysis of RBC from infected poults was 10 to 35% less on day 4 postinoculation (PI) and 50 to 65% less on day 7 PI than that of controls. Red blood cells of poults deprived of water for 3 days were also resistant to lysis; the percent lysis was roughly the same as that of RBC from infected poults at day 7 PI. Incubating control RBC in plasma from infected poults, in extracts of infected' ceca, or at different pH levels did not increase their resistance to lysis, suggesting that neither a stabilizing factor in the plasma that had rapid effect on the RBC nor a transient shift in blood pH was involved. Mean RBC size differed little among infected, water-deprived, and control poults (14.0-14.2 X 8.0-8.1 x 3.8 im). However, although 3.5% of RBC population of control and water-deprived poults were immature (mid to late polychromatic erythrocytes), only 0.4% of the RBC of infected poults were immature. The data suggest that reduced water intake as well as other factors may be involved in the decreased osmotic fragility of RBC from poults infected with E. adenoeides. RESUMEN

Beltsville small white and Nicholas turkey poults, 1, 7, and 14 days old, were inoculated with mixed cultures of Eimeria adenoeides and E. meleagrimitis. Weight gain and feed conversion of 1-day-old poults were affected as severely as those of the older poults, and mortality was heaviest in this group, ranging from 25 to 45%. One dose of oocysts (2 x 10(4)-1.5 x 10(5], given at 1, 7, or 14 days of age, protected even the 1-day-old poults against challenge with 3 x 10(5) oocysts. Protection was comparable to that afforded by multiple immunizing doses given over 3 weeks. After challenge, little weight reduction or mortality was observed in immunized poults. Average feed conversions of the immunized challenged poults were 1.52 to 1.69 as compared with 2.98 to 5.14 for unimmunized challenged poults.

Eimeria adenoeides and E. tenella sporozoites, that were (1) young, unfrozen, (2) young, frozen, or (3) old, frozen, were inoculated into cell cultures of bovine embryonic kidney. At 5 hr, more unfrozen sporozoites were intracellular than were those that had been frozen; of those frozen, more young than old were intracellular. At 48 hr, the percentages of developmental stages from young, frozen sporozoites were (1) similar to those from young, unfrozen sporozoites and (2) greater than those from sporozoites that were old when frozen. Freshly excysted sporozoites of Eimeria meleagrimitis, obtained from oocysts that were 5 to 34 weeks old, survived and developed better in cell culture than those from oocysts that were 54 and 60 weeks old (Doran and Vetterling, 1969b). When freshly excysted or frozen E. meleagrimitis sporozoites of the same age were inoculated into cell culture (Doran, 1969a), more unfrozen than frozen sporozoites were found in cells at 5 hr, but the percentage of development at 48 hr was similar. This report concerns the influence of age and freezing on development of E. adenoeides and E. tenella sporozoites in cell culture. MATERIALS AND METHODS Oocysts were made bacteria-free by treatment with undiluted Clorox (Jackson, 1964). They were stored in Ringer's solution at 3 to 6 C until used. Sporocysts were released from oocysts by grinding (Doran and Vetterling, 1968b) and treated with trypsin-bile solution (Doran and Vetterling, 1967) at 43 C until at least 85% of the sporozoites had excysted. Oocysts from which E. adenoeides sporozoites were obtained for freezing were 4 and 36 weeks old; those of E. tenella, 3 and 54 weeks old. Unfrozen E. adenoeides sporozoites were 4 to 7 weeks old; those of E. tenella, 3 to 5 weeks old. Frozen sporozoites were stored as before (Doran and Vetterling, 1968a, 1969a) for 1 to 4 weeks. They were frozen in 3-ml amounts under the conditions previously found most favorable for sporozoite survival (Doran, 1969b). Each tube contained 2.5 to 3.0 million sporozoites/ml. Cell cultures were of bovine embryonic kidney (BEK). They were on 10by 35-mm cover glasses in 16by 150-mm Leighton tubes and were prepared from a frozen supply of cells that had been serially passed 21 times prior to freezing. Only cultures that were confluent and did not contain Received for publication 20 June 1969. cell aggregates were used. The growth and maintenance media were the same as before (Doran and Vetterling, 1968b). Freshly excysted sporozoites were concentrated by centrifugation and resuspended in 95% Medium 199 with Hank's balanced salt solution + 5% chicken serum. This medium was also used to dilute the suspension of previously frozen sporozoites so that the dimethyl sulfoxide content was less than 1%. The concentration of sporozoites in each of the 3 suspensions was adjusted so that 1.0 ml contained the desired inoculum. As before (Doran, 1969b), only those sporozoites that were refractive to the microscope light were counted. After adjusting the suspending medium to pH 7.0 to 7.2 and thorough agitation, 1.0 ml of each suspension was pipetted into 6 Leighton tubes. In Experiment 1 the inoculum was either 150,000 E. adenoeides or 100,000 E. tenella; in Experiments 2 and 3, 300,000 of either species. At 4 hr, the medium used to inoculate the sporozoites was replaced with 5 ml of maintenance medium (pH 7.0 to 7.2). Cultures were kept in a walk-in incubator with a regulated temperature cycle as previously described (Doran and Vetterling, 1968b). At 5 and 48 hr, cover glasses were removed from 3 tubes in each set of inoculated cultures. They were fixed and stained as before (Doran and Vetterling, 1968b). Counts of sporozoites, trophozoites, and schizonts were made at 645 X. They represent the total number of each found by examining 4 equally spaced "rows" across the length of each of 3 cover glasses. Differentiation of sporozoites as Types A and B was made on the basis of size, intensity of staining, and nuclear morphology. As previously mentioned (Doran and Vetterling, 1968b), a Type B sporozoite was larger, more intensely stained with a nucleus containing a large, prominent nucleolus.