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SYNOPSIS. In young first generation schizonts of E. bovis, the nuclei appeared to have a random distribution. In calves killed 8 days after inoculation some of the schizonts had the nuclei arranged in a single layer at the periphery, with a few infoldings of this layer into the interior. In further development, such ingrowths of the nuclear layer resulted in the formation of compartments of varying size. In schizonts of calves killed 12 days after inoculation spherical or ellipsoidal bodies (blastophores), about 5–20 μ in diameter with a single peripheral layer of nuclei were formed. Merozoites developed as radial outgrowths from the blastophores, leaving residual bodies of variable size, which later disappeared. The response of the host cell to the presence of the schizont was characterized by marked growth of both the nucleus and cytoplasm. The nucleolus became greatly enlarged, and the chromatin was distributed in relatively fine granules. In the host cell cytoplasm, 2 concentric layers were observed; the inner was more dense than the outer. After growth of the schizont was completed its host cell was stretched into a thin covering layer about 1 μ thick. In some schizonts, the host cell disintegrated, and the schizont was then invaded by eosinophils, macrophages and other cells, which eventually destroyed the merozoites.

SYNOPSIS. Eimeria bovis merozoites occurred in tissue culture medium removed from Leighton tube cultures of embryonic bovine tracheal cells beginning 12-14 days after inoculation with 270,000-369,000 sporozoites per tube. The number of merozoites produced in these cultures increased daily until a peak was reached 18-21 days after inoculation. In 3 experiments an average of 2.0–15.6 million merozoites per tube was produced during the 20-day observation period. When such merozoites were frozen in liquid nitrogen and stored 26–42 days, some were motile upon thawing. These merozoites as well as others freshly obtained from cell cultures and from calves were inoculated into 11 different types of cultured mammalian cells including primary, cell line and established cell line cultures. Some merozoites were exposed to substances normally found in the lumen of the gut, before or at the time of inoculation. Altho small numbers of intracellular merozoites were found, no further development was observed. Gametocytes were observed in the cecum of a calf 4 days after merozoites from cell cultures were introduced into a ligated cecum of the calf.

Summary Three experiments were conducted to determine whether schizonts of E. bovis are appreciably influenced by the immune reaction in calves. Each experiment involved two groups each of four to six calves. Each calf in one group was given an immunizing inoculation of 60,000 to 100,000 oocysts, and after 30 to 35 days each calf in both groups was given a challenging inoculation of 800,000 to 5 million oocysts. One calf in each group was killed on successive days beginning 15 to 17 days after the second inoculation. Numbers of schizonts in the small intestine were estimated by examination with a dissecting microscope and frequency of sexual stages in the cecum and colon was observed in sections. No consistent difference was found in concentration, estimated total number, or size of schizonts in the two groups, although non-immunized calves had a much higher frequency of sexual stages. The results indicate that the schizonts and preceding stages are not appreciably influenced by the immune reaction, so that immunity must chiefly affect the merozoites or sexual stages.

To determine whether Eimeria bovis has a second asexual generation occurring in the large intestine of calves, three biopsy experiments were conducted. In each, 0.5 to 1.0 billion E. bovis merozoites were introduced into the ligated cecum of a young calf. The merozoites were in mucosal scrapings obtained from the small intestine of a calf killed 15 days after inoculation with 2.0 million oocysts, 95% E. bovis. During the 6 to 72 hr after the inoculation of merozoites, three or four cecal biopsies were made in each calf. One calf was killed 80 hr after inoculation. The other two discharged only E. bovis oocysts on the 5th and 6th and 5th to 7th day after inoculation, respectively. In the histological sections of the cecum of each calf, many immature schizonts were found in the tissues taken at 6 to 36 hr, numerous mature schizonts were seen at 42 to 48 hr, many very early gametocytes at 48 hr, and progressively more advanced gametocytes at 60, 72, and 80 hr. Mature schizonts in the sections had a mean size of 8.9 by 10.0 t; they contained 30 to 36 merozoites, averaging 1.2 by 3.5 p. Fresh merozoites were 5.7 to 6.8 /L long, with a mean of 6.2. The findings demonstrate that a second asexual generation is included in the life cycle of E. bovis and occurs in the epithelial cells of the cecum and colon. The life cycle of Eimeria bovis was reported by Hammond, Bowman, Davis, and Simms (1946) to include apparently only a single generation of large schizonts occurring in the small intestine. The sexual stages were found to be located in the glands of the cecum and colon. In recent work on the immunology of E. bovis we frequently found small schizonts in the large intestine of calves inoculated with this species. These schizonts typically occurred in association with the gametocytes of E. bovis. For this reason experiments were conducted to determine whether E. bovis has a second generation of schizonts developing in the cecum and colon. Received for publication 28 January 1963. * Supported in part by research grant E-2374 from the Institute of Allergy and Infectious Diseases and by a fellowship (GPM-18,586) from the Division of General Medical Sciences, Public Health Service. The research was conducted under a cooperative agreement between the Utah Agricultural Experiment Station and the Animal Disease and Parasite Research Division, Agricultural Research Service, U. S. Department of Agriculture. Published as Journal Paper No. 306, Utah Agricultural Experiment Station. MATERIALS AND METHODS Six Holstein-Friesian male calves, 3 to 5 weeks old, were used in a series of three biopsy experiments. Three of the six calves were used as sources of merozoites for inoculation. The methods of housing, feeding, inoculating, and sampling for oocysts were similar to those already described (Hammond, Andersen, and Miner, 1963). In each experiment, by means of a laparotomy performed under local anesthesia, the cecum was ligated and a small piece was removed from the blind end for comparison with sections to be obtained after inoculation. After rinsing out the cecum with physiological saline solution the incision was partially closed. Fifty to 60 ml of mucosal scrapings, containing approximately 0.5 to 1.0 billion E. bovis merozoites, were then introduced by syringe, and the suturing of the incision was completed. The mucosal scrapings were obtained from the "source" calves, which were killed 15 days after inoculation with 2.0 million oocysts, 95% E. bovis, 3% E. auburnensis, 1% E. ellipsoidalis, and less than 1% E. cylindrica. The scrapings were collected in saline or Ringer's, concentrated by sedimentation and centrifugation, and homogenized for about 30 sec in a blender to free the merozoites from the schizonts. The interval between the killing of one calf and the introduction of the merozoites into the cecum of another was 3 to 5 hr. The incision in the flank in two of the calves was closed with a temporary suture, through and through, using umbilical tape. In the third calf a row of loops of umbilical tape was placed along 428 This content downloaded from 207.46.13.184 on Sun, 10 Apr 2016 04:38:11 UTC All use subject to http://about.jstor.org/terms HAMMOND ET AL.-LIFE CYCLE OF EIMERIA 429 TABLE I. Results of biopsies in three calves given cecal inoculations with merozoites of E. bovis. Dimensions ( microns) Hr after No. of Chief stages found inoc. biopsies Mean Range 6 1 Schizonts, 1 nucleus 2.0 X 2.0 (3)1 1.8 X 1.8 to 2.2 X 2.2 12 2 Schizonts, 1 nucleus 2.7 X 3.3 (15) 1.3 X 1.9 to 3.8 X 3.8 24 2 Schizonts, 1 to 8 nuclei (mean 4.7) 4.9 X 5.7 (50) 2.9 X 2.9 to 7.2 X 8.6 36 1 Schizonts, 8 nuclei to mature 6.7 X 7.9 (18) 5.7 X 5.7 to 7.2 X 14.3 (mean 15.0) 42 1 Schizonts, 8 nuclei to mature 8.3 X 9.0 (40) 5.7 X 7.2 to 10.0 X 11.4 (mean 19.5) 48 2 Mature schizonts 8.9 X 10.0 (100) 7.2 X 7.2 to 11.4 X 11.4 Very early gametocytes 2.3 X 2.5 (100) 1.3 X 1.9 to 3.2 X 3.2 60 1 Early intermediate gamnetocytes 3.3 X 3.6 (100) 2.6 X 2.6 to 4.5 X 4.5 72 1 Intermediate gametocytes 7.0 X 7.3 (100) 5.1 x 5.1 to 9.6 X 11.5 802 1 Late intermediate gametocytes 11.0 X 12.5 (100) 9.0 X 9.0 to 12.8 X 19.2 1 Number of specimens. 2 Tissues obtained at necropsy instead of biopsy. each side of the incision, and the wound closed by lacing between the loops. Three or four additional cecal biopsies were performed in each of the three experiments at intervals of (no. 1) 6, 12, 24, and 48 hr; (no. 2) 24, 48, and 72 hr; and (no. 3) 12, 36, 42, and 60 hr. The cecal ligature was removed at 24 or 36 hr. The first calf was killed 80 hr after the operation. The other two were allowed to survive so that the discharge of oocysts could be determined; the incision in the flank was permanently closed after the last biopsy in these two calves. At each biopsy a small piece of cecum was removed and bisected; one portion was fixed in Serra's and the other in Helly's or Zenker's. The former tissue was stained with periodic-acid-Schiff and with Hime's and Moriber's methods, the latter with iron hematoxylin and hematoxylin-eosin. Measurements of stained coccidial stages were made with the iron hematoxylin preparations. Observations were also made on mucosal scrapings from fresh cecal tissue.

Thirty-one Holstein-Friesian calves were inoculated with Eimeria auburnensis oocysts; one or more were killed at daily intervals from 4 to 17 days after inoculation. Also, eight calves were infected by the introduction of excysted sporozoites of E. auburnensis into intestinal fistulas, and two calves by placing sporozoites into the intestine via laparotomy. The tissues from calves were prepared by several histological techniques for microscopic examination. First-generation asexual stages were located in epithelial cells at or near the base of the crypts in the jejunum and ileum. Numerous intracellular sporozoites were observed in fistulas of calves killed 4 and 5 days after inoculation. No typically rounded, uninucleate trophozoites were found. In 5-day fistulas, many of the parasites had an appearance similar to that of sporozoites, except that they were larger and had two to six nuclei. Often, such sporozoite-shaped schizonts had lateral bulges with one or more nuclei. This probably represents a stage in the formation of spheroidal schizonts. Early schizonts had nuclei which were randomly arranged; in later schizonts the nuclei assumed a peripheral location. Beginning on the 8th day after inoculation, numerous invaginations of the peripheral nuclear layer of the developing schizont occurred. Each invagination had a lightly staining lumen, which was continuous with the parasitophorous vacuole; each wall bordering the lumen had a single layer of closely spaced nuclei. In schizonts approaching maturity, merozoites were formed by budding from the walls of the invaginations. In mature schizonts, first observed at 10 days after inoculation, the merozoites were randomly distributed throughout the space occupied by the schizont, including the parasitophorous vacuole. Such schizonts contained many thousands of merozoites and occupied the whole width of the crypt, with few or no epithelial cells remaining along the sides. A small amount of glycogen was observed in intracellular sporozoites; more occurred in schizonts, and first-generation merozoites had many granules of glycogen in the middle two-thirds of their bodies. Nuclei of asexual stages were Feulgen-positive. Crescent-shaped bodies of unknown function were associated with 5-day sporozoites and 5and 6-day schizonts. In response to the presence of the parasite, host cell nuclei increased in size; the chromatin became less prominent, and the nucleoli became greatly enlarged. There was no apparent increase in volume of the host cell cytoplasm. Some of the epithelial cells adjacent to the parasitized cells underwent degeneration. Eimeria auburnensis Christensen and Porter, 1939 is a coccidium of cattle having a low degree of pathogenicity as compared with E. bovis and E. zuernii. E. auburnensis is of special interest because the sexual stages are located more deeply in the host tissues than usual, parasitizing cells of the lamina propria, rather than epithelial cells (Hammond, Clark, and Miner, 1961). Little is known about the location and development of the asexual stages in the life cycle of this species. Davis and Bowman (1962) found large schizonts and microgametocytes in the lamina propria near the muscularis mucosae in the small intestine Received for publication 25 October 1968. * Supported in part by NSF Research grant GB5338, and by Public Health Service Fellowship 5-F1-GM-31,285 from the Institute of General Medical Sciences. Published as Journal Paper No. 831, Utah Agricultural Experiment Station. tPresent address: Department of Biology, Andrews University, Berrien Springs, Michigan 49104. of two calves. The relatively large size of the sporozoites (Nyberg and Hammond, 1965; Hammond, Chobotar, and Ernst, 1968) provides a favorable situation for the study of early postinvasion changes in the developing parasites and in host cells. The study reported herein was undertaken to determine the development of the first-generation schizont of E. auburnensis and its relationship to the host cells and tissues. METHODS OF PROCEDURE All calves were purchased locally when less than 1 week old, and housed in individual 5by 10-ft pens constructed of plywood panels. The rear half of each pen was covered with corrugated sheet metal. The floor consisted of a 6-inch layer of coarse gravel, covered with straw, which was changed weekly. At the time of purchase, each calf was given intramuscularly 500,000 international units of vitamin A, 75,000 of vitamin D2, and 50 of vitamin E. Each calf was fed twice daily 2 to 3 pints of raw whole milk mixed with 1 pint of warm water; also, hay and mixed grain were provided. At the beginning of the 4th or 5th week the animals were placed on a diet of

Four experiments were conducted, in each of which 0.4 to 1.9 billion first-generation merozoites of Eimeria bovis were inoculated by laparotomy into the ligated cecum of a nonimmunized calf and a calf immunized with 25,000 or 50,000 oocysts. These merozoites were obtained from calves killed 15 days after inoculation with 2.0 million oocysts. An initial biopsy was performed at the time merozoites were inoculated, and three or four additional biopsies were done in each calf at intervals of 3 to 24 hr during the following 2 or 3 days. The ligature was removed 1 to 2 days after inoculation, and fecal samples collected daily were examined for oocysts. Approximately 1 week after the last biopsy in each experiment, the calves were challenged with an oral inoculation of 1.0 million oocysts and were killed 19 days later. Moderate infection occurred after the inoculation of merozoites in the nonimmunized calves of Experiments 1 and 2, but only slight infections occurred in the nonimmunized calves of Experiments 3 and 4. No infection could be detected in any of the immunized calves, except for a few parasites found in one calf at 24 hr. These findings suggest that the penetration of host cells by first-generation merozoites is inhibited in immunized calves. All of the immunized calves and two of the four nonimmunized calves showed little or no infection after challenge, but the nonimmunized calves of Experiments 3 and 4 became moderately infected. These results indicate that infection caused by inoculation into the cecum of first-generation merozoites stimulates the development of immunity. Although immunity against coccidiosis is well known in both mammals and birds, little has been learned about the way in which the parasites are affected by the immune reaction. The occurrence of unusually large first-generation schizonts in E. bovis, with the resulting ready availability of merozoites, makes this species suitable for investigation of this problem. The present study was undertaken to determine whether first-generation merozoites of E. bovis inoculated into the cecum of immunized and nonimmunized calves would be able to invade the epithelium and initiate an infection, and to determine whether infections so produced would result in immunity. Received for publication 22 January 1964. * Supported in part by research grant E-2374 from the Institute of Allergy and Infectious Diseases and by a fellowship (GPM-18,586) from the Division of General Medical Sciences, Public Health Service. Published as Journal Paper No. 385, Utah Agricultural Experiment Station. t Present address: College of Veterinary Medicine, University of Illinois, Urbana, Illinois. MATERIALS AND METHODS Twelve Holstein-Friesian male calves were used in a series of four biopsy experiments. Four of these calves were used as sources of merozoites for inoculation. The methods of housing, feeding, inoculating, sampling for oocysts, collecting and concentrating merozoites, and performing the biopsies were as described previously (Hammond, Andersen, and Miner, 1963a, b). In each experiment, one calf was immunized by inoculating it when 1 to 2 weeks old with 25,000 or 50,000 E. bovis oocysts. Approximately 1 month later, this animal and a nonimmunized calf of similar age were each given a cecal inoculation with 0.4 to 1.9 billion first-generation merozoites of E. bovis. These merozoites were obtained by scraping the intestinal mucosa of a calf killed 15 days after inoculation with 2.0 million oocysts. More than 90% of these oocysts were E. bovis; the remainder consisted chiefly of E. ellipsoidalis and E. auburnensis. Gametocytes of these two species were seen, in addition to E. bovis schizonts, in the intestinal sections of the calves used as sources of merozoites. However, the cecal inoculations resulted in pure E. bovis infections, as shown by the finding of oocysts of this species only in the fecal samples. In each experiment equal numbers of merozoites were introduced by laparotomy into the ceca of the two calves. Three or four additional biopsies