Generation Schizonts Research Articles

The Absence of Gut Microbiota Alters the Development of the Apicomplexan Parasite Eimeria tenella

Coccidiosis is a widespread intestinal disease of poultry caused by a parasite of the genus Eimeria. Eimeria tenella, is one of the most virulent species that specifically colonizes the caeca, an organ which harbors a rich and complex microbiota. Our objective was to study the impact of the intestinal microbiota on parasite infection and development using an original model of germ-free broilers. We observed that germ-free chickens presented significantly much lower load of oocysts in caecal contents than conventional chickens. This decrease in parasite load was measurable in caecal tissue by RT-qPCR at early time points. Histological analysis revealed the presence of much less first (day 2pi) and second generation schizonts (day 3.5pi) in germ-free chickens than conventional chickens. Indeed, at day 3.5pi, second generation schizonts were respectively immature only in germ-free chickens suggesting a lengthening of the asexual phase of the parasite in the absence of microbiota. Accordingly to the consequence of this lengthening, a delay in specific gamete gene expressions, and a reduction of gamete detection by histological analysis in caeca of germ-free chickens were observed. These differences in parasite load might result from an initial reduction of the excystation efficiency of the parasite in the gut of germ-free chickens. However, as bile salts involved in the excystation step led to an even higher excystation efficiency in germ-free compared to conventional chickens, this result could not explain the difference in parasite load. Interestingly, when we shunted the excystation step in vivo by infecting chickens with sporozoites using the cloacal route of inoculation, parasite invasion was similar in germ-free and in conventional chickens but still resulted in significantly lower parasite load in germ-free chickens at day 7pi. Overall, these data highlighted that the absence of intestinal microbiota alters E. tenella replication. Strategies to modulate the microbiota and/or its metabolites could therefore be an alternative approach to limit the negative impact of coccidiosis in poultry.

Gross and Histopathology of Coccidiosis in Small Ruminants in Tamil Nadu

Coccidiosis, caused by protozoa of the genus Eimeria, is one of the major parasitic diseases characterized by subclinical contagious enteritis in domestic and wild animals. A total of 125 tissue samples (Abomasum, small intestine and large intestine) of sheep and goat were collected from slaughter houses in Chennai, Kanchipuram and Tiruvallur districts and samples from necropsied small ruminants at Department of Veterinary Pathology, Madras Veterinary College, Chennai, Tamil Nadu, India. The overall prevalence of coccidiosis in single infection in collected samples was 28.57% (10/35). Grossly, the ileum and jejunum infected with coccidia showed oedema, congestion of serosa and mucosa. In few cases, the ileum showed a single white round raised spot. On histopathological examination, infected tissues showed different developmental stages like early, young and mature first generation schizonts with stage of blastophore formation, early stage of compartmentalization, advanced stage of compartmentalization, early and mature second generation schizonts with stage of merozoite formation, macromeronts, micromeronts, mature meronts, microgametocytes, macrogametocytes, microgamonts, macrogamonts and mature oocysts in the intestinal mucosa mainly in the epithelial crypts.

Re-Evaluation of Asynchronous Asexual Development of Cystoisospora canis in Intestines of Dogs

The coccidian parasite Cystoisospora canis (syn. Isospora canis) can cause clinical disease in dogs. Three generation of schizonts have been reported in the small intestine of dogs before oocysts are excreted 9-11 days post inoculation (PI). Here, we re-evaluated asexual development of C. canis in 2 dogs necropsied 10 days after oral inoculation with 100,000 C. canis oocysts; both dogs had excreted oocysts 9 days PI. Asexual and sexual stages were seen in the lamina propria throughout the small intestine. Merozoites of different sizes were present, often in the same vacuole. They were arranged singly, in pairs, and many within a single parasitophorous vacuole. The maximum number of nuclei within developing merozoites in a group was 8, but it could not be discerned if they were individual nuclei or parts of merozoites. Findings of abundant asexual stages 1 day after dogs had started excreting oocysts indicated continued asexual multiplication beyond the prepatent period. The stages found resemble the 3 generations reported previously. The mode of division of the asexual generations remains unclear. The results of the present study indicate that there are many generations that are difficult to determine because of the multiplication of merozoites in the original host cell without leaving it to enter new host cells. From the literature, it is evident that cat and dog coccidia ( Cystoisospora spp.) divide by more than 1 type of division, including endodyogeny. In the past, the schizont/meront groups containing more than 1 generation have been called "cysts." However, cyst is not an accurate term because it is best used for an orally infective stage of coccidia; monozoic tissue cysts of C. canis can occur in paratenic hosts in extraintestinal organs. We recommend the term "types" as originally proposed for intestinal stages of Toxoplasma gondii and used for the original description of the life cycle of C. suis of swine when describing endogenous stages of the Sarcocystidae. Ultrastructural studies are needed to determine the precise form of multiplication of canine Cystoisospora species.

Artemisinin and Artemisia annua leaves alleviate Eimeria tenella infection by facilitating apoptosis of host cells and suppressing inflammatory response

Evasion strategies of intracellular parasites by hijacking cellular pathways, are necessary to ensure successful survival and replication. Eimeria tenella (E. tenella) has the ability to circumvent apoptosis of infected cells through increased expression of the transcriptional factor NF-κB and the anti-apoptotic factor Bcl-xL during the development of second generation schizonts. Artemisinin (ART) and its original plant, the dried leaves of Artemisia annua (LAA) have been shown to be effective against avian coccidiosis, however, the underlying mechanism remains unclear. We showed that E. tenella infection promoted the expression of anti-apoptotic protein Bcl-2 and inhibited the expression of pro-apoptotic proteins Bax and cleaved caspase-3 at 60 h post infection (PI), with a higher ratio of Bcl-2 to Bax. Nevertheless, the expression trends of Bcl-2, Bax and caspase-3 were reversed at 120 h and 192 h PI. ART treatment significantly abrogated Bcl-2 expression, whereas it promoted the expression levels of Bax and cleaved caspase-3 at the three time points above. Additionally, ART remarkably suppressed the increased mRNA expressions of NF-κB and interleukin-17A in ceca during infection by E. tenella. Compared with the ART treatment, LAA treatment exerted more improvements in clinical symptoms, promoting apoptosis and suppressing inflammatory response. These alterations caused by ART and LAA treatments were consistent with the reduced clinical diarrhea and pathological improvements in chicken ceca. Collectively, these results indicate that the inhibitory effects of ART or LAA on E. tenella infection may work through facilitating the apoptosis of infected host cells and inhibiting the inflammatory response.

Histopathological Studies of Eimeria bovis Infection in Calves

Six experimentally infected calves were slaughtered at 15 days post infection (dpi) (Group A), 20 dpi (Group B), 25 dpi (Group C) and the intestinal mucosa was examined for pathological changes. The affected part of the intestine was removed, opened and examined for macroscopic findings. The animals studied showed lesions of varying severity. The gross pathology revealed no remarkable abnormal findings in calves of group A. Redness of the mucosa of the caecum and colon in six calves of group B and C was noted. There were no first generation schizonts evident grossly. The main characteristic under the light of microscope of the intestinal sections taken from the calves examined during the late prepatent period were mild inflammatory infilteration of the mucosa and increased cellular debris, parasitic structures were identified from small and large intestine. The caecum and colon showed particularly high levels of infection.

Electron microscopic study of the developmental stages of Eimeria intestinalis cheissin, 1948 in domestic rabbit. (Oryctolagus Cuniculus) with reference to endodyogeny.

The endogenous stages in Eimeria intestinalis were studied in experimentally infected coccidia-free rabbits by transmission electron microscopy. Four asexual generations were ob- served.Two types of merozoites were reported. Binucleated merozoites possess all the characteristics of apical complex indicating asexual reproduction by endodyogeny. While the second type, the mononuclear merozoites have one central nucleus. Gamonts were developed mainly from the third generation merozoites where the mature gamonts were recorded together with the fourth generation schizonts. Macro- and microgametogenesis were clearly observed. Development of microgametes and its fine structural characteristics were detected. Mature macrogametes with central large nucleus and the appearance of two types of wall forming body (I, II) were observed. Many reserve food materials including amylopectin granules and lipids droplets were very characteristic. Control experimentally infected rabbits shed unsporulated oocysts on the day eight p.i.

Localization of Eimeripain, an <i>Eimeria tenella</i> Cathepsin B-Like Cysteine Protease, during Asexual and Sexual Intracellular Development in Chicken Ceca

ABSTRACTHemorrhagic diarrhea in poultry is caused by Eimeria tenella, the most pathogenic avian coccidian parasite, and new approaches to treat the disease are continually being sought. Although eimeripain, a cathepsin B-like cysteine protease from E. tenella, has recently been identified as a novel anticoccidial drug target, its localization during the intracellular development of parasites remains unclear. Here, we demonstrate the expression of eimeripain during asexual and sexual development of E. tenella in vivo. Promature eimeripain was detected only in the early immature second generation of schizonts. In contrast, the mature eimeripain was most strongly detected in the middle-sized immature second generation of schizonts. Both promature and mature eimeripain disappeared depending on the maturation level of second generation of schizonts, but were strongly expressed again in the third generation of schizonts. In the sexual stage, both promature and mature eimeripain were detected in the cytoplasm of micro- and macro-gametocytes and zygotes, but expression became weak in zoites forming oocysts. Collectively, our findings suggest that eimeripain might play a key role in the differentiation of intracellular zoites in the ceca and could be an interesting candidate to develop a novel, effective anti-coccidian drug.

LIGHT AND ELECTRON MICROSCOPIC STUDY OF MEROGONY OF EIMERIA SP. INFECTING THE MARBLED TEALS (ANAS ANGUSTIROSTRIS)

Two asexual generations were developed before the commencement of the sexual phase. First generation schizont containing 12-16 randomly scattered merozoites masured 19.5 x 16.3 um, supranuclear and ectomerogonic with residual body. Each merozoite being vermiform and measured 7.7 x 2.4 um. The merozoites formation was completed within 48 hours. post-infection (p.i). Meanwhile, mature second generation schizont measured 17.5 x 15 um. It conained 4-20 merozoites, each measured 9.5 x 2.8 um, and the process was completed within 84 hours p.i. The present study showed all changes in the ultrustructural forms associated with the development, starting from uninuclear schizont to multinuclear schizont. The process of merozoite formation, recorded in the present study, termed ectomerogony in which the merozoites developed at the periphery of each schizont. This method is common and recorded among many species of Eimeria. The electron microscope study revealed that the pellicle consists of two membranes, an outer thin unit membrane and an inner complex one usually formed of two membranes. During this stage, the merozoites of the first generation invaded new uninfected epithelial cells. The same events, recorded in the formation of the first generation schizonts and merozoites were carried out in the second generation, except that the second has specific arrangement of merozoites, and it was rich with endoplasmic reticulum. The outer pellicular unit membrane of merozoites was continous enclosing the whole merozoite without interruptions except an invagination at the site of micropore.

Effect of ochratoxin A on coccidiosis-challenged broiler chicks

A study was conducted to evaluate the effects of ochratoxin A (OTA) on broiler chicks challenged with Eimeria tenella oocysts. Two hundred day-old, unsexed Cobb broiler chicks were randomly divided into four treatment groups. Each treatment consisted of five replicates and ten chicks per replicate, making the following treatments: group I: control; group II: OTA (1 mg/kg) daily through feed; group III: coccidia (orally inoculated with 50,000 E. tenella oocysts/chick on day 21); group IV: OTA (1 mg/kg) daily through feed + coccidia (orally inoculated with 50,000 E. tenella oocysts/chick on day 21). Six birds from each group were slaughtered on the 5th, 7th, 9th and 11th day post infection. The results showed higher mortality with severe gross lesions in caecum and a greater number of faecal oocysts in groups III and IV. The gross lesions observed in group IV were characterised by distension of caecum with blood-tinged content indicative of haemorrhagic typhlitis with mucosal tissue debris. Microscopically, lymphoid organs revealed severe lymphocytolysis and depletion with cellular sparsity in OTA treated groups. The increased severity of lesions in the caecum of group IV was attributed to the additive effect of OTA and E. tenella. Caecum exhibited severe haemorrhages, the presence of numerous second generation schizonts, matured merozoites and developing oocysts. Group IV showed an increase in the severity of coccidiosis which is due to the immunosuppressive effect of OTA. Thus, it was concluded that the expression of E. tenella and its pathological effects were maximum in the presence of OTA compared to the incidence of coccidiosis alone in broiler chicks.

Development of Eimeria bovis in vitro: suitability of several bovine, human and porcine endothelial cell lines, bovine fetal gastrointestinal, Madin–Darby bovine kidney (MDBK) and African green monkey kidney (VERO) cells

Several bovine, human and porcine endothelial cell lines, bovine fetal gastrointestinal cells (BFGC), Madin-Darby bovine kidney (MDBK) and African green monkey kidney (VERO) cells were exposed in vitro to sporozoites of Eimeria bovis. Parasites invaded all cells used and changed their shape to more stumpy forms within 12 h. Sporozoites left their host cells and invaded new ones frequently within the first 12 h post-infection. Further development took place only in bovine cells, although parasites survived in the other cells for at least 3 weeks. Within the non-bovine cells, conspicuously enlarged parasitophorous vacuoles developed in VERO cells and reached a diameter of 15-20 microm. The best development to first generation schizonts with regard both to time required to mature, to schizont size and to merozoite yields was observed in BFGC, followed by bovine umbilical vein and bovine spleen lymphatic endothelial cells. MDBK cells were less suitable. The life cycle was completed (development of oocysts) only occasionally in BFGC. Results are considered under several aspects. Thus, infected VERO cells may represent a suitable tool for studying the parasitophorous vacuole, while infected endothelial cells represent a system quite narrow to the in vivo situation and should allow basic studies on parasite/host cell interactions and BFGC can be used for the mass production of E. bovis first generation merozoites.

Pathogenesis of Eimeria colchici in the Intestine of Chickens and their Immune Response

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Immunity in rats against Eimeria separata: oocyst excretion, effects on endogenous stages and local tissue response after primary and challenge infections.

The aim of the study was the characterization of the local immune response of Lewis rats to Eimeria separata, a caecum-dwelling coccidium. Rats infected twice at 10-day intervals with 5,000 oocysts developed a high degree of immunity to a heavy challenge with 100,000 oocysts, reducing the oocyst production by > 98% when compared with naive recipients. Histopathological investigations performed over a period of 0-72 h post-infection (pi) showed that 1st generation schizonts, developed within 24 h pi, represented the major target stages, although later stages were also affected. Preinfected animals showed significantly more lymphocytes in the caecum wall than naive animals. An increase in lymphocyte numbers after challenge observed in both groups was enhanced in challenged animals up to 36 h pi. The number of lamina propria lymphocytes predominantly was increased after primary infection whereas in repeatedly infected animals the increase also concerned intraepithelial lymphocytes. In addition, the numbers of plasma cells were enhanced in the caecum wall of immune animals. Macrophage infiltration in the caecum wall followed a similar time course in both groups up to 36 h pi. A subsequent further rise up to 48 h pi was enhanced in naive rats. Tissue infiltrations with eosinophils and mast cells were observed predominantly in the repeatedly infected rats. No obvious changes occurred with intestinal neutrophils and goblet cells. In conclusion, caecum tissue alterations suggest an early local immune response, which is related to development and maturation of the parasite.

Life-cycle of Isospora mehlhornii sp. nov. (Apicomplexa : Eimeriidae), parasite of the Egyptian swallow Hirundo rubicola savignii.

Fifty-seven Hirundo rubicola savignii swallows were collected from Damietta, Tanta, Dakahlyia and Sharkia Provinces, Egypt. They were examined for coccidian parasites. The percentage of infection with Isospora stages was 12.3%. After diagnosis it was noted that the parasites belong to a new species. The unsporulated oocysts were spherical, measuring 25.7-31.9 microm in diameter with a mean of 28.3 microm. A micropyle, polar granule and oocyst residuum were absent. Sporocysts appeared lemon-shaped and measured 19.6-24.5 microm x 12.5-17.5 microm with a mean of 22.9 x 14.4 microm. Stieda body and the sporocyst's residual body were clearly visible. Sporozoites measured 11-14.2 x 4.4-5.1 microm with a mean of 11.4 x 4.7 microm. Sporulation time was 72 h at room temperature (25 degrees C). Endogenous stages including schizogony and gamogony were detected in epithelial cells of the duodenum, jejunum and ileum of the host. Schizogony consisted of two generations. Mature first generation schizonts reached up to 11 microm in diameter and produced merozoites measuring 3.5 x 1.7 microm. Mature second generation schizonts measured 17.2 x 12.3 microm and produced merozoites measuring 11.8 x 2.2 microm. Gamogonic stages were differentiated into microgamonts and macrogamonts. Mature microgamonts were spherical and measured 23.2 microm in diameter, producing curved microgametes measuring 4.5 x 0.7 microm. The ovoid macrogametes measured 19.6 x 14.7 microm and were characterized by a large nucleus and nucleolus. Early, more or less spherical, oocysts were detected inside the intestinal epithelial cells and in the intestinal lumen. They measured 19.6 microm in diameter. The sporont measured 17.2 microm in diameter. Cytochemical studies on schizogony, gamogony and oocysts were accomplished and showed distribution of polysaccharides and composition of the oocyst wall.

T cell responses in calves to a primary Eimeria bovis infection: phenotypical and functional changes

The study aimed to characterize T cell responses in calves to a primary E. bovis infection. For this purpose, peripheral blood lymphocytes (PBL) were isolated from six infected calves and three controls during prepatency (Day 12 post infection (p.i.), patency (Day 25 p.i.) and postpatency (Day 35 p.i.). In addition, lymphocytes were isolated from various lymphatic organs ( lnn. cervicales superficiales, lnn. jejunales craniales, lnn. jejunales caudales, lnn. caecales, lnn. colici, Peyer’s patches (PP) and spleen) at necropsy (Day 35 p.i.). FACS analyses determined the proportions of CD4 +-, CD8 +-, CD2 +-, and γδ +-T cells. Proliferative responses of the cells after stimulation with Concanavalin A (Con A) and an E. bovis-merozoite I antigen (EbAg) were measured. Furthermore, in situ hybridization experiments were performed for the detection of IL-2 and IL-4 mRNA in histological sections of lymphatic organs. Proportions of CD4 +-, CD8 +- and CD2 +-expressing PBL were significantly increased 12 days p.i. in infected calves. While the proportions of CD4 +- and CD8 +-PBL declined until day 25 p.i. and finally reached control values, proportions of activated PBL (CD2 +-T cells) remained at a high level throughout the observation period. Those of γδ +-PBL, in contrast, remained unaffected. The proportions of CD4 +-, γδ +- and CD2 +- T cells in lymphatic organs were significantly increased in comparison to uninfected controls, when determined 35 days p.i. Concerning the proportions of CD8 +-T cells of the organs, however, there were no differences between the groups. PBL and cells from lymphatic organs except those from the PP showed strong proliferative response to the mitogen Con A, without a significant difference between the groups. Reactions to EbAg in contrast differed significantly between controls and E. bovis infected calves. Proliferation responses of PBL of infected animals were highest 12 days p.i.; subsequently they decreased and 35 days p.i. they were found within the ranges of controls. Lymphocytes isolated from lymphatic organs of infected animals reacted significantly stronger than lymphocytes from control animals, whereby most marked differences occured with cells from lymph nodes draining E. bovis infested parts of the intestine and from the spleen. These reactions were accompained by an increased transcription of the IL-2 gene but not of the IL-4 gene in gut associated lymphnodes of infected calves when compared with infected controls. The data suggest strong antigenic stimuli by developing first generation schizonts, and of predominant involvement of (CD4 +) Th1 cells in the course of a primary E. bovis infection of calves.

Eimeria tenella: cloning and characterization of cDNA encoding a S3a ribosomal protein.

A lambda Zap II cDNA library was constructed from Eimeria tenella first- generation schizonts mRNA and screened with a mouse serum raised against this parasitic stage. This serum identified a clone encoding a S3a ribosomal protein (EtS3a). The 858-bp cDNA fragment, containing the entire parasitic gene encoded a highly basic protein of 264 amino acids (aa) with a molecular weight of 29.780kDa. Based upon amino acid sequence comparison, EtS3a is highly homologous to v-fos transformation effector (encoded by the fte-1 gene) and cyc-07 (a plant homologue of fte-1) and similar to the yeast MFT1 (encoded by the mitochondrial fusion targeting gene). The expressions of mammalian fte-1, plant cyc-07 and yeast MFT1 have all been shown to be cell-cycle-regulated and involved in protein synthesis at the level of the ribosome. Since EtS3a expression is also developmentally regulated, we suggest that this gene product is a functional homologue of fte-1, cyc-07 and MFT1 and an important molecule regulating the development of Eimeria tenella.

Transient expression of β-galactosidase in differentiating sporozoites of Eimeria tenella

A transient transfection system has been developed for a member of the Apicomplexa, Eimeria tenella, using β-galactosidase ( βgal) from Escherichia coli as the reporter enzyme. Successfully expressed constructs contained sequences of the E. tenella microneme gene Etmic-1 fused to the coding region of lacZ. Transfectants expressing βgal were able to invade host cells and proceed through part of the life-cycle, forming schizonts from which merozoites were released. This indicated that transfectants could differentiate at least to first generation schizonts. However, this differentiation was delayed compared with unelectroporated sporozoites by approximately 15 h. Some merozoites arising from transfected sporozoites also expressed βgal. These results are encouraging for the development of a stable transfection system for E. tenella, using βgal as a reporter enzyme.

Selection and development of a Spanish precocious strain of Eimeria necatrix

A precocious line of Eimeria necatrix (PEN E-281/20) with an abbreviated life cycle was derived from a Spanish field strain (E-281) by repeated passages of the first shed oocysts recovered from the caecal contents of previously infected chickens. After 20 passages, the `useful' prepatent period (time from infection to obtaining sufficient oocysts to repassage) of the parasite was reduced by 30 h (from 148 to 118 h). The earliest oocysts found in the caecal content were 114 h postinfection (hpi), on the 19th passage. The pathogenicity of the parasite was reduced in comparison with the parent strain, its immunogenicity against homologous and heterologous strains was maintained and its reproductive capacity was similar to or higher than that of the parent strain. Compared with the parent strain, the second generation of schizonts was reduced in size (reduced pathogenicity), third generation schizonts were bigger and with more merozoites (maintenance of the reproductive index) and the life cycle progressed faster from the second generation of schizonts (reduction of prepatent period). Complete second schizogony, from trophozoites to mature schizonts was observed frequently in the caeca of birds infected with both parent and precocious lines.

Monoclonal antibodies inhibiting invasion of cultured cells by Eimeria tenella sporozoites.

Monoclonal antibodies (MoAbs) secreted by 12 hybridomas and reactive with the surface antigens of E. tenella sporozoites were produced. These MoAbs designated as KC-1 to KC-12 were characterized as IgG3 kappa. In Western blot analysis, these MoAbs reacted with only one polypeptide band of sporozoite antigens having a molecular mass of 25 kDa. All the MoAbs were reactive with sporozoites, trophozoites, immature and mature first generation schizonts, first generation merozoites, and the interior structure of sporulating oocysts of E. tenella, however, not with any of the methanol-fixed sporozoites of E. acervulina, E. mitis, E. maxima, E. brunetti, E. necatrix and E. praecox. Invasion of primary culture of chicken kidney cells by E. tenella sporozoites was significantly inhibited by several of the MoAbs. These results suggest that these MoAbs recognizing the E. tenella-specific surface molecule are involved in the invasion of sporozoites into host cells.

Protective immunity induced with the antigens fractionated by affinity chromatography from the second generation schizonts of Leucocytozoon caulleryi.

A crude extract of the 2nd generation schizonts (2GS) of Leucocytozoon caulleryi and chicken serum containing serum-soluble antigens were applied to an immunoaffinity column using immunoglobulin of chickens recovered from L. caulleryi infections. The fractionated antibody-bound proteins showed several bands between 70 kDa and 35 kDa by the electrophoretic analysis. By immunization of chickens, the fractionated proteins conferred the protection at varying degrees comparable to that conferred by the unfractionated original materials. These results indicated that antiserum against 2GS can recognize the proteins involved in protective immunity.

Confocal Laser Scanning Microscopy of β-Tubulin and α-Actinin in Asexual Stages of Eimeria tenella (Apicomplexa: Eimeriidae) in Cell Culture

Summary: Specific monoclonal antibodies were used to visualize the dynamics of β-tubulin and α-actinin in asexual development of Eimeria tenella in cultured primary chicken kidney cells by means of confocal laser scanning microscopy. β-tubulin was more condensed in refractile bodies (RB), at anterior ends, and along subpellicles in sporozoites. The RB remained labelled during the development of first generation schizonts until the formation of merozoites, suggesting a possible important role of RB in the reorganization of microtubules in schizonts. The microtubules also formed a number of tiny aggregation centers scattered throughout the schizonts corresponding to the formation of first generation merozoites. The microtubular material in second generation schizonts was clustered around several weakly-stained areas. Both first and second generation merozoites had more highly concentrated β-tubulin at anterior ends and along subpellicles. In contrast, α-actinin, and actin-binding protein, was more concentrated in the anterior half area of sporozoites. It became evenly distribution in the whole cytosol of developing trophozoites except RB areas. The distribution of α-actinin in first or second generation schizonts corresponded to merozoite formation. The β-tubulin and α-actinin of chicken primary kidney cells were also labelled by the two monoclonal antibodies.