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Chicks fed betaine supplemented diets and infected with Eimeria acervulina and Eimeria maxima had markedly higher levels of betaine in the duodenum and mid-gut than unsupplemented, infected chicks. Uninfected chicks fed betaine exhibited almost twice the levels of betaine in the gut as infected chicks. Plasma betaine levels were lower in E. maxima-infected chicks than in E. acervulina-or Eimeria tenella-infected chicks. Betaine supplementation reversed the decrease in weight gain in E. maxima- infected chicks but had no effect on the decrease in weight gains in E acervulina- and E. tenella-infected chicks. Coccidia-infected birds on normal diets regularly exhibit increases in plasma NO(2)(+)NO(3). This increase was abolished in E.tenella-infected birds on betaine supplement. Betaine feeding did not alter this effect in E. acervulina- and E. maxima-infected birds. Results indicate that betaine supplementation has a positive effect on gut betaine levels in birds infected with E. acervulina and E. maxima. In all treatment groups, infection lowered the levels of betaine.

The effects of polyclonal antisera to 2 recombinant dense granule (DG) antigens of Neospora caninum on invasion and development by N. caninum tachyzoites in baby hamster kidney cell cultures were examined. In immunofluorescent antibody tests, the anti-DG sera, at dilutions of 1:100 to 1:500, reacted intensely with individual intracellular tachyzoites and groups of tachyzoites enclosed in parasitophorous vacuoles at 2 and 52 h p.i., respectively. Tachyzoites suspended in diluted anti-DG sera and inoculated immediately into the cell cultures invaded cells in significantly fewer numbers (53–68% fewer by 2 h p.i.) than tachyzoites suspended in similarly diluted normal rabbit serum. In contrast, tachyzoites suspended in anti-Eimeria tenella sporozoite serum invaded cells as efficiently as those suspended in normal rabbit serum. Addition of anti-DG sera at the time of inoculation of tachyzoites, or to the cell cultures after the parasites had entered cells, had little effect on either the percentage or rate of their subsequent development.

At 7 d postinoculation (DPI) with a mixed culture of avian Eimeria species, 21-d-old chicks maintained in batteries and floor pens on a diet containing 0.15% (3 lb/ton) betaine plus 66 ppm (60 g/ton) salinomycin were significantly heavier and had significantly lower feed conversion ratios and mortality than chicks fed diets containing 0.15% betaine or 66 ppm salinomycin alone, or the control diet. At 31 DPI, when the chicks were 45 d old, the differences between the diet groups were not as great as at 7 DPI. In vitro, except at high concentrations, betaine was nontoxic to sporozoites of Eimeria tenella or Eimeria acervulina and had little effect on their invasion and development in cultured cells. In vivo, invasion by E. tenella and E. acervulina sporozoites was significantly reduced in all chicks fed diets containing betaine or salinomycin compared with that in control chicks. There was a significant interaction between betaine and salinomycin that impacted on invasion by both species. Overall development of E. tenella did not appear to be adversely affected by addition of betaine to diets containing salinomycin. Conversely, development of E. acervulina was reduced in chicks fed diets containing 0.075% (1.5 lb/ton) betaine plus 66 ppm salinomycin as compared with that in chicks fed salinomycin alone.

The 8th International Coccidiosis Conference, held on 9--13 July 2001 in Palm Cove, Australia, was a showcase of the latest studies on widely known coccidia, including Eimeria and Toxoplasma in addition to the emerging or re-emerging parasites such as Neospora, Cryptosporidium and Cyclospora. This meeting was staged in conjunction with the Annual Scientific Meeting of the Australian Society for Parasitology.

The effect of conditioned media from cultures of turkey and chicken intestinal cells on cellular invasion by sporozoites of avian Eimeria species was examined in vitro. Media conditioned by the growth of cells from the ceca, mid-intestine (area of the yolk stalk diverticulum), and duodenal loop were examined for their ability to enhance invasion. Conditioned medium from cultures of turkey cecal cells significantly enhanced invasion by the turkey coccidia Eimeria adenoeides, by 2.4-fold, and E. meleagrimitis, by 2.2-fold, as compared with invasion in the presence of control medium. Conditioned medium from mid-intestinal cell cultures enhanced invasion by the two coccidial species by 2.0- and 2.1-fold, respectively. The enhancement occurred with conditioned media from early (1) as well as later (11) passages of cells. This suggests that the enhancing factor was produced by fibroblast-like cells, the predominant cell type at both early and late passages, and not by epithelial-like cells that had disappeared by the first or second passage. Additionally, conditioned media from cultures of chicken cecal and duodenal loop cells significantly enhanced invasion by the turkey cecal coccidium, E. adenoeides, (1.7- and 1.6-fold, respectively). This was less enhancement than was caused by the turkey cell conditioned media. Heat treatment (56 degrees C for 45 min) of conditioned media failed to alter the effect on invasion. Neither the turkey or chicken cecal cell media nor conditioned media from any other chicken intestinal cell cultures enhanced invasion by E. tenella, the chicken cecal coccidium. Although morphologically dissimilar when they were first plated, the gross appearance and growth of the turkey and chicken cells when conditioned media was collected was comparable.

To assess muscle breakdown during avian coccidiosis, the level of the non-metabolizable amino acid 3-methylhistidine (3MH) was determined in muscle and plasma from turkey poults that received an infection with a field isolate containing a mixture of Eimeria species. The effect of increased levels of parasitism was evaluated at 6 days postinoculation (DPI) in birds receiving 2.5 x 10(4), 1 x 10(5), or 2 x 10(5) oocysts each. The changes in 3MH levels during recovery from acute infection were assessed at 6-29 DPI in animals given 1.9 x 10(5) oocysts per bird. In some experiments, uninoculated birds given the same amount of feed as infected birds (pair fed) were used to determine the impact of feed deprivation on weight loss and 3MH levels. Infected birds had significantly elevated plasma and muscle 3MH at 6 DPI after a single dose of Eimeria oocysts. The plasma and muscle 3MH returned to control levels after 14 DPI. The 3MH levels increased with increased dose of oocysts. Plasma and muscle 3MH levels were well correlated, and an inverse curvilinear relationship between weight gain and plasma 3MH concentrations levels was observed. Plasma and muscle 3MH levels were significantly elevated in pair-fed birds, but 3MH levels in infected birds were increased by 30% over pair-fed birds. The results suggested that muscle breakdown, as assessed by plasma and muscle levels of 3MH, increased during the acute stage of Eimeria infection in turkey poults.

The site specificity that avian Eimeria sporozoites and, to a more limited degree, other apicomplexan parasites exhibit for invasion in vivo suggests that specific interactions between the sporozoites and the target host cells may mediate the invasion process. Although sporozoite motility and structural and secreted antigens appear to provide the mechanisms for propelling the sporozoite into the host cell,there is a growing body of evidence that the host cell provides characteristics by which the sporozoites recognise and interact with the host cell as a prelude to invasion. Molecules on the surface of cells in the intestinal epithelium, that act as receptor or recognition sites for sporozoite invasion, may be included among these characteristics. The existence of receptor molecules for invasion by apicomplexan parasites was suggested by in vitro studies in which parasite invasion was inhibited in cultured cells that were treated with a variety of substances designed to selectively alter the host cell membrane. These substance included cationic compounds or molecules, enzymes that cleave specific linkages, protease inhibitors, monoclonal antibodies, etc. More specific evidence for the presence of receptors was provided by the binding of parasite antigens to specific host cell surface molecules. Analyses of host cells have implicated 22, 31, and 37 kDa antigens, surface membrane glycoconjugates,conserved epitopes of host cells and sporozoites, etc., but no treatment that perturbs these putative receptors has completely inhibited invasion of the cells by parasites. Regardless of the mechanism,sporozoites of the avian Eimeria also invade the same specific sites in foreign host birds that they invade in the natural host. Thus, site specificity for invasion may be a response to characteristics of the intestine that are shared by a number of hosts rather than to a unique trait of the natural host. Protective immunity elicited against avian Eimeria species is not manifested in a total blockade of parasite invasion. In fact, the effect of immunity on invasion differs according to the eliciting species and depends upon the area of the intestine that is invaded. Immunity produced against caecal species of avian Eimeria, for example Eimeria tenella and Eimeria adenoeides, inhibits subsequent invasion by homologous or heterologous challenge species, regardless of the area of the intestine that the challenge species invade. Conversely, in birds immunised with upper intestinal species, Eimeria acervulina and Eimeria meleagrimitis, invasion by challenge species is not decreased and often is significantly increased.

The effects of prior (immunity) or concurrent administration of Eimeria acervulina or Eimeria tenella on cellular invasion in vivo and in vitro and on growth performance in white leghorn chickens (WLC) were examined. Weight gains of WLC immunized with E. acervulina and challenged with E. tenella were significantly greater than those of nonimmunized chicks challenged with E. tenella (this occurred despite the increased invasion by E. tenella in E. acervulina-immunized chicks that was reported earlier). The weight gains and modest but consistent improvements in intestinal lesion scores, feed conversion ratios, and oocyst shedding in immunized/challenged WLC indicated that E. acervulina conferred a small measure of protection against E. tenella infection that was independent of the effect on invasion. In contrast, immunization of WLC with E. tenella significantly decreased (41%-51%) invasion by E. acervulina as compared with that in nonimmunized WLC but had little effect on chick growth performance. Concurrent inoculation of chicks with E. tenella and E. acervulina had little effect on invasion by E. tenella sporozoites or on subsequent performance of the chicks. In vitro, prior exposure of cultured cells to either of two isolates of E. tenella also caused a significant decrease in invasion by E. acervulina. No gross changes occurred in the culture morphology between the E. tenella-inoculated and noninoculated cultures. Collectively, the data indicate that prior exposure of WLC and cultured cells to single isolates of avian coccidia markedly influenced invasion by other species but had less effect on the growth performance of the birds.

The effect of betaine and salinomycin on absorption of methionine and glucose in tissue from the duodenal loops of Eimeria acervulina-infected chicks was determined. Differences in the ultrastructure of the intestinal cells and parasite developmental stages were also examined. With a drug-resistant isolate of E. acervulina, methionine absorption was significantly higher in chicks fed a basal diet supplemented with 0.15% betaine as compared with absorption in chicks fed the unsupplemented basal diet. Addition of 66 ppm salinomycin to the diet containing betaine did not further enhance absorption. Conversely, with a drug-sensitive isolate, methionine absorption was significantly higher in chicks fed a diet supplemented with both betaine and salinomycin than in chicks fed the unsupplemented basal diet. Tissue from chicks fed any of the supplemented diets was usually significantly heavier than that from chicks fed the unsupplemented diet, even when weight gains of the birds were similar. Glucose absorption was similar in all diet groups. Epithelial cells in coccidia-infected and uninfected chicks fed diets supplemented with betaine or betaine plus salinomycin were less electron dense than cells from chicks fed diets that were not supplemented with betaine. Merozoites of E. acervulina in chicks fed diets supplemented with salinomycin had extensive membrane disruption and vacuolization, but the damage was prevented when betaine was added to the diet. Numerous merozoites and intact schizonts were seen in the intestinal lumen of chicks fed the diet containing betaine plus salinomycin.

During the past several years, our laboratory has been investigating the anticoccidial activities of various natural products that have potential use as dietary supplements for coccidiosis control. Sources of fats containing high concentrations of n-3 fatty acids such as menhaden oil and flaxseed oil and flaxseed, when added to starter rations and fed to chicks from one day of age, effectively reduce lesions caused by the caecal parasite Eimeria tenella, but not lesions caused by Eimeria maxima. Our results are consistent with reports of effects of diets high in n-3 fatty acids on other protozoan parasites which suggest that the state of oxidative stress induced by these diets in the cells of both host and parasites is responsible for their parasitic actions. Artemisinin, a naturally occurring (Artemisia annua) endoperoxide and effective antimalarial significantly lowers lesions from E. tenella when given at low levels as a feed additive. The mechanism of its action is also considered to involve induction of oxidative stress. Diets supplemented with 8 p.p.m. gamma-tocopherol (abundant in flaxseeds) or with 1% of the spice tumeric, reduce mid-small intestinal lesion scores and improve weight gains during E. maxima infections. These compounds may exert their anticoccidial activity because they are effective antioxidants. Betaine, a choline analogue found in high concentrations in sugar beets, improves nutrient utilisation by animals under stress. When provided as a dietary supplement at a level of 0.15% it has enhanced the anticoccidial activity of the ionophore, salinomycin. Betaine may act as an osmoprotectant whereby it improves the integrity and function of the infected intestinal mucosa. In in vivo studies, betaine plus salinomycin significantly inhibit invasion of both E. tenella and E. acervulina. However, subsequent development of E. acervulina is inhibited more effectively with this combination treatment than development of E. tenella.

Eimeria gruis and E. reichenowi are common coccidial parasites of a number of crane species. In the present study, monoclonal antibodies (McAbs), elicited against Eimeria spp. of chickens and turkeys, cross-reacted with sporozoites and developmental stages of E. gruis in the tissues of Florida sandhill cranes. These McAbs were used to define the area of the intestine that was invaded by sporozoites of E. gruis and to demonstrate the feasibility of using McAbs to study the early development of E. gruis in the intestines and visceral organs of cranes. At 6 hr postinoculation (PI), E. gruis sporozoites were found primarily from just proximal to Meckle's diverticulum in the jejunum to the ileocecal juncture. Fewer sporozoites were found in the ceca and rectum, and none were found in the duodenum. Most of the sporozoites were in the middle third of the villi and within the lamina propria. At 14 days PI, developmental stages were detected in the ceca, jejunum, liver, and lungs but not in the heart, kidney, or brain. In the ceca and jejunum, the number, location, and maturity of the stages differed markedly.

The effect of prior or simultaneous administration of Eimeria acervulina on cellular invasion by E. acervulina, E. tenella, or E. adenoeides was examined in vivo and/or in vitro. In chickens immunized with E. acervulina oocysts, invasion by sporozoites from a challenge inoculum of E. acervulina increased slightly over invasion in naive controls, and invasion by sporozoites of E. tenella and E. adenoeides increased by factors of 1.99 and 2.26, respectively, over that in controls. Conversely, simultaneous inoculation of E. acervulina along with E. tenella or E. adenoeides did not have a significant effect on invasion. Cultured baby hamster kidney cells that had been previously inoculated with E. acervulina sporozoites also supported significantly greater (by factors of 1.39 to 2.66) invasion by E. adenoeides sporozoites than did control cultures. The enhancement of invasion in vitro appeared to depend upon either the interval between the inoculation of cells with E. acervulina and E. adenoeides or the interval between the replating of the cells and their inoculation with E. adenoeides. There were no gross changes in the culture morphology between the E. acervulina-inoculated and noninoculated cultures. The data indicate that prior exposure to E. acervulina enhances invasion by other species of avian coccidia and, therefore, may exacerbate the infection produced by these species.

Repeated oral inoculation of turkey poults with large doses (1 x 10(6) oocysts) of the chicken coccidia, Eimeria tenella or E. acervulina, failed to prevent weight loss, poor feed conversion, and intestinal pathology in turkeys challenged with the turkey coccidium, E. adenoeides. Invasion by E. tenella in turkeys was significantly greater than invasion by E. adenoeides in chickens; by 24 hr postinoculation (PI), the numbers of E. tenella and E. adenoeides sporozoites in the ceca had decreased markedly as compared with the numbers that initially invaded, and they did not differ significantly from each other. At 24 hr PI, however, transfer of cecal scrapings from chickens or turkeys inoculated with E. adenoeides produced infection in 53% of the recipient turkeys, but transfer of scrapings from either chickens or turkeys inoculated with E. tenella failed to produce infection in 20 attempts with recipient chickens. Cultured chicken peripheral blood monocytes (PBMs) that were inoculated with E. adenoeides sporozoites contained numerous vesicles that were recognized by the refractile body-specific monoclonal antibody 1209; the number of vesicles was markedly decreased in PBM cultures inoculated with gamma-irradiated E. adenoeides sporozoites. Very few vesicles were detected in the cytoplasm of turkey PBMs that contained E. tenella sporozoites, and none were detected in turkey PBMs containing E. adenoeides sporozoites. The survival of infective sporozoites, along with the secretion of refractile body antigen, may be more critical to the development of cross-species immunity than the number of sporozoites that initially invade the foreign host.

Unimmunized chickens, given intraperitoneal injections of Sephadex at the same time that they were inoculated with oocysts of either Eimeria tenella or E. acervulina, had significantly lower lesion scores at 6 days postinoculation (PI) than unimmunized chickens that were injected with saline instead of Sephadex. Despite the difference in lesion scores, there was little effect on weight gain, except in one experiment, in which Sephadex-injected chickens gained significantly more weight than saline-injected chickens. In contrast, in chickens that were immunized by prolonged exposure to sporozoites of E. adenoeides, injection of Sephadex at the time of challenge with E. tenella did not reduce lesion scores or parasite development as compared with the uninjected chickens, and the weight gain of the Sephadex-injected challenged chickens fell to a level significantly lower than that of their saline-injected challenged counterparts. The data indicate that Sephadex injected at the time of oocyst inoculation 1) produces markedly different effects on lesion scores and weight gain in unimmunized and immunized chickens and 2) abrogates sporozoite-elicited immunity against E. tenella challenge.

Repeated inoculation (immunization) of white leghorn chickens with oocysts of the turkey coccidium, Eimeria adenoeides, resulted in significantly improved weight gain and feed-conversion ratios (feed efficiency) after E. tenella challenge. However, the development E. tenella in the immunized chickens did not differ markedly from that in unimmunized chickens, and intestinal lesions remained severe (greater than 3.0). Apparently improved weight gain and feed efficiency can be maintained in the presence of extensive parasite development. The ability to elicit cross-protective immunity was abrogated when the E. adenoeides oocysts were exposed to 15 kRad of gamma irradiation before inoculation into the chickens. Sporozoites existing from irradiated oocysts of E. acervulina also failed to immunize the chickens against challenge with E. tenella but protected chickens almost completely against homologous challenge with E. acervulina. The results indicate that cross-species immunity is not elicited by all developmentally arrested sporozoites and that the ability to produce cross-species immunity can be destroyed by gamma irradiation of the immunizing species.

Eimeria maxima oocysts were exposed to various doses of gamma radiation that did not affect sporozoite invasion of intestinal epithelium but did prevent subsequent merogonic development therein. Although merogony and oocyst formation were inhibited, parasites exposed to 12 kRad radiation induced a level of immunity against E. maxima challenge equivalent to that induced by non-irradiated oocysts. Chickens immunized per os with 20 kRad-treated E. maxima oocysts were not protected against coccidial challenge. Immunization of chickens with a single low dose (five oocysts) of non-irradiated (0 kRad) or irradiated (12 kRad) E. maxima was effective in preventing weight depression after coccidial challenge. Immunofluorescence staining of intestinal tissue from chickens infected with irradiated (12 or 20 kRad) or non-irradiated (0 kRad) E. maxima oocysts with developmental stage-specific monoclonal antibodies showed that sporozoite invasion was similar in all groups. However, merogonic development was not observed at any time postinfection in chickens infected with irradiated oocysts, unlike the case with chickens infected with non-irradiated parasites. These results suggest that sporozoite-infected host cells are capable of eliciting complete protection against E. maxima challenge.

In the present study, we wished to demonstrate the ability of surface gametocyte antigens to induce protective immunity against Eimeria maxima infections in chickens. In order to accomplish this goal, we employed maternal immunization as a means of providing large amounts of specific antibodies to offspring chicks. Upon challenge with sporulated E. maxima oocysts, chicks from hens immunized with affinity-purified gametocyte antigens showed greatly reduced oocyst production compared with chicks from sham-immunized hens. These results suggest that maternal immunization with gametocyte antigens can be used as a means to provide transmission-blocking immunity against E. maxima infections.

Sporulated oocysts of the protozoan parasite Eimeria tenella were attenuated by exposure to various doses of X-radiation to inhibit intracellular replication and thus determine whether sporozoites alone can induce a protective immune response. Exposure to doses greater than 15-kilorads had a significant effect on development, as indicated by the absence of oocyst production in chickens infected with parasites treated with 20 or 30 kilorads of radiation. Infection with nonirradiated or 15-kilorad-exposed parasites led to either normal or reduced oocyst shedding. Equivalent protection was afforded chickens inoculated with a minimum immunizing dose of either nonirradiated or 20-kilorad-irradiated E. tenella oocysts. Immunofluorescence staining of cecal tissue from chickens inoculated with 10(7) nonirradiated or 20- or 30-kilorad-irradiated oocysts with stage-specific monoclonal antibodies showed no significant difference in sporozoite invasion between treatment groups. Normal merogonic development was observed at appropriate times (48, 60, 72, and 96 h) postinfection in chickens inoculated with nonirradiated oocysts. In contrast, irradiated parasites exhibited minimal merogonic development at 48 h postinfection. Furthermore, no merogonic stages were observed at times of otherwise peak merozoite development (60, 72, and 96 h) in cecal tissue from chickens inoculated with irradiated parasites. Infection of chicken cells with irradiated or nonirradiated parasites in vitro corroborated these findings and indicate that events early after sporozoite invasion induce a protective immune response against this parasite.

Previous studies showed that molecules of in vitro-cultured primary turkey kidney cells bound to 23-, 40-, and 60- to 65-kDa antigens of sodium dodecyl sulfate-solubilized sporozoites of Eimeria adenoeides. Similar binding to antigens of three other species of avian Eimeria, E. tenella, E. acervulina, and E. meleagrimitis, is now reported. Strips containing the most avidly bound sporozoite antigen (approximately 40 kDa) were excised from the sodium dodecyl sulfate-polyacrylamide gels on which E. adenoeides antigens had been electrophoretically separated. The strips were homogenized and injected into mice to produce hybridoma cell lines. Twelve cell lines secreting monoclonal antibodies (McAb) that reacted with E. adenoeides sporozoites were detected. One of these McAb, H11C3, reacted with structures in the anterior tip of sporozoites of E. adenoeides and five other species of avian Eimeria. When included in the inoculation medium, this McAb significantly inhibited invasion of cultured kidney cells by sporozoites of E. adenoeides and E. tenella. In contrast, when the sporozoites were pretreated with McAb H11C3 and then washed free of the antibody, no inhibition of invasion was observed.

Repeated inoculation (immunization) of 2-week-old white leghorn chickens with 10(6) oocysts of the turkey coccidia Eimeria adenoeides or E. meleagrimitis partially protected chickens against moderate challenge with E. tenella or E. acervulina oocysts, but not with E. necatrix oocysts. After challenge, mean weight gains of the immunized chickens and the unchallenged controls did not differ significantly, but weight gains of unimmunized chickens were significantly lower. The mean feed-conversion ratio of the immunized challenged chickens was 3.14, as compared with 4.42 for unimmunized challenged control chickens. In general, immunization did not markedly reduce intestinal lesions. Repeated inoculation of chickens with the turkey coccidium E. gallopavonis failed to produce statistically significant protection against challenge with E. tenella, E. acervulina, or E. necatrix, as determined by weight gain, feed-conversion efficiency, and lesion scores. Antibody profiles of individual chickens did not correlate with protection.

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.

Sporulated oocysts of the protozoan Eimeria acervulina were subjected to 0, 10, 15, 20, or 30 krad of X-irradiation and inoculated into susceptible outbred chickens to determine if radioattenuated coccidia could induce protection against parasite challenge. Irradiation treatment had an appreciable dose-dependent effect on parasite development. Insignificant numbers of oocysts were produced by chickens inoculated with parasites that had been exposed to greater than 10 krad X-irradiation. Sporozoites exposed to 15 or 20 krad irradiation conferred significant protection against the appearance of intestinal lesions after parasite challenge. Sporozoites subjected to the highest dose level (30 krad) did not produce any significant level of protection. To investigate this phenomenon further and assess intracellular parasite development, susceptible outbred strains of chickens were administered either nonirradiated (0 krad) oocysts or oocysts that were exposed to an optimal dose (15 krad) or a high dose (30 krad) of X-irradiation. Immunofluorescence staining of tissue sections from each treatment group at various intervals after the initial administration of irradiated parasites indicated that sporozoites exposed to 15 krad irradiation were as capable of invading the host intestinal epithelium as nonirradiated sporozoites. However, at 48, 60, 72, and 96 hr, there was a marked reduction in merogonic developmentmore » in groups receiving irradiated sporozoites compared to those inoculated with nonirradiated parasites. The latter parasites underwent profuse merogonic development; in contrast, irradiated parasites demonstrated little (15 krad) or no (30 krad) merogonic development. These results suggest that induction of a protective immune response occurs during a critical period early in intracellular development of E. acervulina.« less

Four species of avian Eimeria invaded the intestine of foreign host birds in the same areas in which they invaded the natural host. Repeated inoculation (immunization) of chickens with the turkey coccidian, Eimeria adenoeides, partially protected the chickens against a subsequent challenge with 5.8 x 10(4) E. tenella oocysts. At 6 days post-challenge, the weight gain and feed conversion efficiency of the immunized chickens was significantly better than those of the chickens that were not immunized with E. adenoeides. Lesion scores and cellular invasion by the sporozoites were significantly lower in the immunized birds than in the unimmunized group. Electrophoresis and Western blot analysis identified changes in the serum antibody profiles of the chickens that appeared to be associated with the immunization and challenge programs. An antibody or antibodies recognizing a 60,000-molecular-weight antigen of E. tenella sporozoites disappeared when chickens immunized with E. adenoeides were challenged with E. tenella; an antibody or antibodies recognizing a 23,000-molecular-weight sporozoite antigen appeared within 6 days of challenge. Reciprocal studies, in which turkeys were immunized with E. tenella and challenged with E. adenoeides, showed little evidence of protection.

To assess muscle breakdown during avian coccidiosis, the level of the non-metabolizable amino acid 3-methylhistidine (3MH) was determined in muscle and plasma from turkey poults that received an infection with a field isolate containing a mixture of Eimeria species. The effect of increased levels of parasitism was evaluated at 6 days postinoculation (DPI) in birds receiving 2.5 x 10(4), 1 x 10(5), or 2 x 10(5) oocysts each. The changes in 3MH levels during recovery from acute infection were assessed at 6-29 DPI in animals given 1.9 x 10(5) oocysts per bird. In some experiments, uninoculated birds given the same amount of feed as infected birds (pair fed) were used to determine the impact of feed deprivation on weight loss and 3MH levels. Infected birds had significantly elevated plasma and muscle 3MH at 6 DPI after a single dose of Eimeria oocysts. The plasma and muscle 3MH returned to control levels after 14 DPI. The 3MH levels increased with increased dose of oocysts. Plasma and muscle 3MH levels were well correlated, and an inverse curvilinear relationship between weight gain and plasma 3MH concentrations levels was observed. Plasma and muscle 3MH levels were significantly elevated in pair-fed birds, but 3MH levels in infected birds were increased by 30% over pair-fed birds. The results suggested that muscle breakdown, as assessed by plasma and muscle levels of 3MH, increased during the acute stage of Eimeria infection in turkey poults.

Eimeria maxima oocysts were exposed to various doses of gamma radiation that did not affect sporozoite invasion of intestinal epithelium but did prevent subsequent merogonic development therein. Although merogony and oocyst formation were inhibited, parasites exposed to 12 kRad radiation induced a level of immunity against E. maxima challenge equivalent to that induced by non-irradiated oocysts. Chickens immunized per os with 20 kRad-treated E. maxima oocysts were not protected against coccidial challenge. Immunization of chickens with a single low dose (five oocysts) of non-irradiated (0 kRad) or irradiated (12 kRad) E. maxima was effective in preventing weight depression after coccidial challenge. Immunofluorescence staining of intestinal tissue from chickens infected with irradiated (12 or 20 kRad) or non-irradiated (0 kRad) E. maxima oocysts with developmental stage-specific monoclonal antibodies showed that sporozoite invasion was similar in all groups. However, merogonic development was not observed at any time postinfection in chickens infected with irradiated oocysts, unlike the case with chickens infected with non-irradiated parasites. These results suggest that sporozoite-infected host cells are capable of eliciting complete protection against E. maxima challenge.

SUMMARY. Four species of avian Eimeria invaded the intestine of foreign host birds in the same areas in which they invaded the natural host. Repeated inoculation (immunization) of chickens with the turkey coccidian, Eimeria adenoeides, partially protected the chickens against a subsequent challenge with 5.8 x 104 E. tenella oocysts. At 6 days post-challenge, the weight gain and feed conversion efficiency of the immunized chickens was significantly better than those of the chickens that were not immunized with E. adenoeides. Lesion scores and cellular invasion by the sporozoites were significantly lower in the immunized birds than in the unimmunized group. Electrophoresis and Western blot analysis identified changes in the serum antibody profiles of the chickens that appeared to be associated with the immunization and challenge programs. An antibody or antibodies recognizing a 60,000-molecularweight antigen of E. tenella sporozoites disappeared when chickens immunized with E. adenoeides were challenged with E. tenella; an antibody or antibodies recognizing a 23,000molecular-weight sporozoite antigen appeared within 6 days of challenge. Reciprocal studies, in which turkeys were immunized with E. tenella and challenged with E. adenoeides, showed little evidence of protection.

Developmental stages of Eimeria meleagrimitis Tyzzer were found throughout the intestine and ceca of turkeys given inocula ranging from 10(4) to 7.5 x 10(5) sporulated oocysts/bird. Infection initially occurred in the duodenum and upper jejunum but later moved down the intestine and into the ceca. The speed with which the infection moved into these areas was rougly proportional to the inoculum size. Heaviest infections were in the ileum, neck of the cecum, and large intestine. The life cycle consisted of 5 asexual generations before gametogony, a 6th asexual generation developing simultaneously with gametogony. First- and 2nd-generations were located along the sides of villi in the upper intestine rather than in the crypts of Lieberkuhn, as previously described in England for this species. Transitory first-generation stages that were abnormally large and usually degenerate were found in the neck of the cecum.

Prophylactic levels of three ionophorous antibiotics, monensin, salinomycin, and lasalocid, were administered to groups of chickens and turkeys. All three ionophores markedly inhibited invasion of cecal tissues by sporozoites of ionophore-sensitive (IS) Eimeria tenella. Monensin and salinomycin also reduced invasion in turkeys by sporozoites of E. adenoeides, but lasalocid only minimally inhibited invasion. Invasion of ceca of monensin-medicated chickens was significantly greater by sporozoites of ionophore-resistant (IR) E. tenella than of the IS isolate. Concomitant experiments showed significant differences in [14 C]monensin accumulation among IS and IR isolates of E. tenella. The decreased uptake of monensin by the IR isolates appeared to be accompanied by a decrease in responsiveness to the activity of monensin as well as to two other ionophores, salinomycin and narasin in cell culture. The amount of monensin, salinomycin or narasin required to inhibit development of E. tenella by 50% was 20 to 40 times higher for the IR isolates than for the IS ones. Collectively, the data suggest that differences in ionophore accumulation by IS and IR isolates of E. tenella might reflect differences in membrane chemistry and that these differences are responsible for the expressions of resistance that were observed in these studies. This expression of resistance appears to be common to all ionophores tested.

SYNOPSIS. Sporozoites and 1st-, 2nd-, and 3rd-generation merozoites of Eimeria meleagrimitis were inoculated into primary cultures of turkey kidney cells. In vitro-excysted sporozoites developed into mature macrogamonts in 8 days; in vivo-excysted sporozoites developed into 2nd- or 3rd-generation schizonts within 5 to 7 days. First-generation merozoites obtained from infected turkeys produced mature 2nd-generation schizonts within 24 h. Second-generation merozoites from turkeys produced mature macrogamonts and oocysts within 72 h, whereas 3rd-generation merozoites produced these stages within 48 h. The oocysts that developed from 3rd-generation merozoites sporulated at 25 C and were infective for turkeys. The timing of the early stages and the intervals between schizogonic generations in cultures were comparable with those in turkeys. Morphologic parameters, however, indicated that some differences existed between in vitro and in vivo development. Second- and 3rd-generation schizonts and gamonts that developed after inoculation of cultures with merozoites were similar to stages in turkeys. Oocysts, however, were significantly smaller (P < 0.05) in cultures. All stages that developed after inoculation of cultures with sporozoites were smaller (P < 0.05) than their in vivo counter parts.

Weight and biochemical studies were conducted on 2-week-old turkeys inoculated with 10(4) to 5 X 10(5) sporulated Eimeria meleagrimitis oocysts, on their pair-fed controls (equivalent food intake), and on control turkeys fed ad libitum. Food consumption and rate of weight gain of all inoculated and pair-fed turkeys fell sharply on day 4 postinoculation (PI), but deaths occurred primarily among the birds inoculated with 5 X 10(5) oocysts. Heart weights (expressed as percentage of body weight) were reduced in inoculated and pair-fed birds, but liver, spleen, and pancreas weights did not differ from those of either control group. Feed conversion (feed consumed/gain) was less efficient for inoculated turkeys than for ad libitum or pair-fed controls and was least efficient for turkeys inoculated with 5 X 10(5) oocysts. Plasma glutamic oxaloacetic transaminase (GOT, aspartate and aminotransferase) activity increased, and carotenoid and total protein levels decreased in inoculated turkeys but not in the pair-fed turkeys, indicating that these changes were caused by the infection and not by reduced food intake. Plasma glutamic pyruvic transaminase (GPT, alanine aminotransferase) remained stable in all groups. Plasma glucose levels of inoculated birds did not differ from those of the control groups, but liver glucose and glycogen levels decreased in both the inoculated and pair-fed birds.

A fusion protein of β-galactosidase and Eimeria tenella produced in a recombinant Escherichia coli strain was injected into chickens and elicited partial protection against an oral challenge with Eim. tenella parasites. The fusion protein contained a 31 kilodalton (kD) coccidial antigen designated as 5401. The DNA sequencing of the 5401 antigen-coding sequence revealed that this protein segment was highly negatively charged and strongly hydrophilic, and contained an amino-acid sequence repeated five times. A dose-titration study showed that immunizing chickens with a single subcutaneous injection of the 5401 antigen at 1,200 to 4,800 nanograms (ng)/bird in Freund's complete adjuvant decreased lesion scores, mortality, and feed conversions compared to unimmunized, challenged controls. Using the 1,200 and 2,400 ng/bird of the 5401 antigen, group weight gains were higher than for the unimmunized, challenged birds. In three other trials using the 5401 antigen at 2,400 ng/bird with light, medium, and heavy coccidial infections, significant protection was evidenced by reduced lesion scores, increased individual weight gains, or both. In addition, feed conversions were reduced when compared with unimmunized controls or birds immunized with a noncoccidial protein E. coli extract. Western blot analysis of sporozoite preparations with serum from 5401-immunized birds labeled two antigenic bands of 66 and less than 200 kD. These results indicate that the coccidial proteins produced in E. coli are potentially effective immunogens for protecting chickens against avian coccidiosis.

The osmotic fragility of red blood cells (RBC) from Eimeria adenoeides-infected turkey poults was compared with that of RBC from control and water-deprived poults. At different hypotonic NaCl 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 microns). 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.

Eimeria dispersa (turkey strain) and Eimeria gallopavonis sporozoites were inoculated into primary cultures of chicken kidney (CK) and turkey kidney (TK) cells. Eimeria dispersa sporozoites were more infective in either cell type than those of E. gallopavonis: at 4 hr, the percentage of infection was 67-98 for E. dispersa but only 23-56 for E. gallopavonis. E. dispersa also survived better in culture: at 2 days, losses of E. dispersa in both cell types were only 4-19%, whereas losses of E. gallopavonis were 35-47% IN TK cells and 60-95% CK cells. However, E. gallopavonis developed further than E. dispersa. Location and increase in numbers of intracellular stages at 4 days indicated that E. dispersa proceeded through 2 schizogonic generations before development stopped.

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.

Developmental stages of Eimeria meleagrimitis Tyzzer were found throughout the intestine and ceca of turkeys given inocula ranging from 10(4) to 7.5 x 10(5) sporulated oocysts/bird. Infection initially occurred in the duodenum and upper jejunum but later moved down the intestine and into the ceca. The speed with which the infection moved into these areas was rougly proportional to the inoculum size. Heaviest infections were in the ileum, neck of the cecum, and large intestine. The life cycle consisted of 5 asexual generations before gametogony, a 6th asexual generation developing simultaneously with gametogony. First- and 2nd-generations were located along the sides of villi in the upper intestine rather than in the crypts of Lieberkühn, as previously described in England for this species. Transitory first-generation stages that were abnormally large and usually degenerate were found in the neck of the cecum.

Physiologic characteristics were measured on day 7 postinoculation (PI) in 2-week-old turkeys inoculated with 10(5) Eimeria adenoeides oocysts, in pair-fed controls, and in control turkeys fed ad libitum. Pathophysiologic responses were measured in turkeys inoculated with 7 x 10(4) E. adenoeides oocysts or 1.2 x 10(5) 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 that 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 PI 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.

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.

Two-wk-old broiler chicks were inoculated via crop intubation with Eimeria acervulina at two doses: 10(5) or 10(6) sporulated oocysts/bird or with Eimeria tenella at a dose of 10(5) sporulated oocysts/bird. Serum and liver samples were collected on days 3 and 6 post-inoculation (PI). There were no significant changes in serum or liver zinc, copper, and iron concentrations in any of the infected groups by 3 d PI. However, on d 6, PI serum protein was significantly reduced in all of the infected groups compared to their pair-fed controls. The chicks infected with E. tenella had significantly reduced serum zinc (1.20 vs 1.77 micrograms/mL) and iron (0.44 vs 1.28 micrograms/mL) concentrations and significantly elevated serum copper (0.28 vs 0.17 micrograms/mL) and ceruloplasmin levels (20.33 vs 11.11 micrograms/mL) compared to their pair-fed counterparts. Those chicks infected with E. acervulina (10(6) oocysts/bird) exhibited significantly reduced serum iron concentration by 6 days PI (0.90 vs 1.14 micrograms/mL). Liver zinc was significantly increased in the chicks infected with E. tenella (349 vs 113 micrograms/g dry liver wt), as was copper (24 vs 19 micrograms/g), whereas liver iron concentration was significantly reduced (172 vs 243 micrograms/g) compared to pair-fed controls. At both dose levels, the chicks infected with E. acervulina exhibited a significant reduction in liver iron by 6 d PI. Hepatic cytosol metals generally reflected whole tissue levels. Metallothionein (MT)-bound zinc was significantly elevated in the chicks infected with E. tenella. Iron bound to a high molecular weight, heat-stable protein fraction (presumably cytoplasmic ferritin) was significantly reduced in chicks infected with E. acervulina, as well as those infected with E. tenella. Collectively, the changes in serum zinc, copper, and iron concentrations, as well as the changes in hepatic zinc and MT-zinc concentrations in the chicks infected with E. tenella were similar to changes evoked during an acute phase response to infection. It is possible that a secondary bacterial infection or inflammation stemming from erosion of the lining of the cecum may play a role in the response of trace element metabolism to the E. tenella infection.

Glucose and methionine were malabsorbed in some intestinal regions of turkeys infected with Eimeria meleagrimitis, E. adenoeides , or E. dispersa . The decrease in absorption was not always related to the numbers of parasites in the cells or the extent of damage to the mucosa. With E. adenoeides , malabsorption was found in the jejunum even though parasites were not present. Conversely, with E. dispersa , no malabsorption was observed in the duodenum even though light microscopy showed numerous parasites. In many intestinal regions, damage to the mucosal surface visible with scanning electron microscopy (SEM) was slight or absent, although malabsorption was marked. No changes were noted with SEM in the structure and orientation of the brush border in these regions. Villar height was significantly reduced in the regions of heaviest infection when intestinal damage was visible. Conversely, the crypts of Lieberkuhn were often two or three times as deep in infected poults as in uninfected poults. In general, no differences were found in the thickness of the circular and longitudinal muscle layers between the infected and uninfected poults. The dry weight of the intestinal tissue was less from infected poults than from uninoculated controls and was related to both region of the intestine and severity of the infection.

The electrophoretic distribution of serum proteins was measured in chickens inoculated with sporulated oocysts of Eimeria acervulina, E. tenella, and E. maxima. Total serum protein decreased on day 5 and 7 postinoculation (PI) with all three species. However, the changes produced in the individual serum protein fractions differed with the species. In serum from E. acervulina-infected birds, all protein components decreased quantitatively; the largest decrease was in the alpha globulins. The overall percent distribution of the globulins was similar to that of uninoculated controls, except for a decrease in the alpha globulins. In E. tenella-infected chickens, albumin decreased (both quantitatively and in percent distribution), but the alpha 1, beta, and gamma 1 globulins increased. In E. maxima-infected chickens, only albumin and alpha 1 globulin decreased quantitatively; the amounts of alpha 2, beta, or gamma globulins did not change. These changes were transitory and had disappeared by day 10 PI.

Heart composition and function were measured in turkey poults inoculated with Eimeria meleagrimitis or E. adenoeides oocysts. Heart weights of infected poults were significantly lower than those of uninfected controls. Lipid levels of the heart were significantly reduced on day 4 postinoculation (PI) with E. meleagrimitis and on day 6 PI with E. adenoeides. The reduced lipid levels were closely associated with decreases in body weight. The protein per gram of heart tissue increased as the lipid levels decreased. Heart glycogen, when expressed as microgram per milligram of protein, decreased. However, the concentration of glycogen per gram of tissue apparently changed very little. Moisture levels of hearts from infected and control poults did not differ significantly. Heart rates and mean blood pressures of poults infected with E. adenoeides were slightly (approximately 8%) lower than values in control poults. However, exposure to stress in the form of ice baths or intravenous injection of epinephrine caused more severe and prolonged fluctuations in heart rate and blood pressure of the infected poults than in those of the controls.

The invasion of the intestinal epithelium of immunized and unimmunized turkeys and chickens by four species of Eimeria was quantitated. In unimmunized birds, E. adenoeides, E. acervulina, and E. tenella invaded primarily the areas in which first-generation schizonts subsequently developed. Eimeria meleagrimitis invaded a larger area of the intestine. Between 1 and 4 hr postinoculation, the numbers of intracellular sporozoites increased, but their location within the intestine was little changed. When birds were immunized with either of two lower intestinal species, E. adenoeides or E. tenella, and then challenged with the immunizing species, invasion was reduced by 36% to 55%. In contrast, immunizing and then challenging birds with either of two upper intestinal species, E. meleagrimitis or E. acervulina, did not reduce invasion: there were 44% more intracellular sporozoites in E. meleagrimitis-immunized turkeys and 11% more in E. acervulina-immunized chickens than in their unimmunized counterparts.