Second-generation Schizonts Research Articles

Anticoccidial evaluation of a traditional chinese medicine—Brucea javanica—in broilers

The traditional Chinese medicinal plant Brucea javanica has received much attention for its significant antiprotozoal effects in recent years; however, little is known about its potential anticoccidial functions. In the present study, a series of experiments was conducted to investigate the prophylactic and therapeutic effects of ethanol extract from B. javanica on coccidiosis induced by Eimeria tenella in broiler chickens. Chickens infected with E. tenella were treated with B. javanica extract and compared either with broilers treated with the anticoccidial halofuginone hydrobromide (Stenorol) or with control groups that consisted of infected-unmedicated and uninfected-unmedicated broilers. The experiments revealed that the B. javanica extract could significantly (P<0.05) reduce bloody diarrhea and lesion scores. Additional, OPG output in these plant extract treated groups was reduced in comparison with non-treated groups (P<0.05). However, there was no evidence to show that the extract could promote BWG. Histological data showed that the number of second-generation schizonts in the medicated groups was substantially less than that in the infected-unmedicated control. In summary, our work showed that B. javanica extract exerted considerable anticoccidial effects, supporting its use as a promising therapeutic in controlling avian coccidiosis.

Ultrastructural effects of acetamizuril on endogenous phases of Eimeria tenella.

To explore the primary stage or site of action of acetamizuril (AZL), a novel triazine anticoccidial compound, the ultrastructural development of Eimeria tenella at different endogenous stages was studied in experimentally infected chickens treated with a single oral dose of 15 mg/kg AZL. As a result of drug action, the differentiations of second-generation schizonts and microgamonts were largely inhibited and merozoites became irregular in shape. Meanwhile, the outer membrane blistering and perinuclear space enlargement were obvious in the second-generation schizonts and microgamonts, which were never observed in the classic triazine anticoccidiosis drug diclazuril-treated E. tenella. The chromatin aggregation, anachromasis, and marginalization were visible in merozoites and microgamonts. Furthermore, the abnormal evagination of microgametes finally resulted in the degeneration of microgamonts and the failure of subsequent fertilization. The most marked micromorphological alteration occurring in the macrogamonts was the WFB2. Even if the fertilization occurred, the formation of oocyst wall became malformed and the zygote proceeded to the obvious degeneration. In addition, mitochondria swelling and cytoplasm vacuolization were discerned in respective intracellular stages, while endoplasmic reticulum and Golgi body swelling was less seen. These alterations may be the causes leading to the final death of E. tenella and also provide some information for further exploring the mechanism of action of AZL at the molecular level.

IL-17A regulates Eimeria tenella schizont maturation and migration in avian coccidiosis

Although IL17A is associated with the immunological control of various infectious diseases, its role in host response to Eimeria infections is not well understood. In an effort to better dissect the role of IL17A in host-pathogen interactions in avian coccidiosis, a neutralizing antibody (Ab) to chicken IL17A was used to counteract IL17A bioactivity in vivo. Chickens infected with Eimeria tenella and treated intravenously with IL17A Ab, exhibited reduced intracellular schizont and merozoite development, diminished lesion score, compared with untreated controls. Immunohistological evaluation of cecal lesions in the parasitized tissues indicated reduced migration and maturation of second-generation schizonts and reduced lesions in lamina propria and submucosa. In contrast, untreated and infected chickens had epithelial cells harboring second-generation schizonts, which extend into the submucosa through muscularis mucosa disruptions, maturing into second generation merozoites. Furthermore, IL17A Ab treatment was associated with increased parameters of Th1 immunity (IL2- and IFNγ- producing cells), reduced levels of reactive oxygen species (ROS), and diminished levels of serum matrix metalloproteinase-9 (MMP-9). Finally, schizonts from untreated and infected chickens expressed S100, Wiskott-Aldrich syndrome protein family member 3 (WASF3), and heat shock protein-70 (HSP70) proteins as merozoites matured, whereas the expression of these proteins was absent in IL17A Ab-treated chickens. These results provide the first evidence that the administration of an IL17A neutralizing Ab to E. tenella-infected chickens inhibits the migration of parasitized epithelial cells, markedly reduces the production of ROS and MMP-9, and decreases cecal lesions, suggesting that IL17A might be a potential therapeutic target for coccidiosis control.

Booster responses by oral vaccination with transgenic plants against chicken leucocytozoonosis

We developed a transgenic potato (TrP/R7) expressing the recombinant R7 (rR7) antigen for use as an oral vaccine to protect against a chicken protozoan disease, chicken leucocytozoonosis. The TrP/R7 potato was produced by Agrobacterium tumefaciens-mediated transformation and regeneration, and the R7 gene insertion into potato chromosomes was confirmed by genomic polymerase chain reaction and Southern hybridization. rR7 antigen expression in TrP/R7 potato was also confirmed by sandwich enzyme-linked immunosorbent assay and western blotting using an antibody against the second-generation schizont of Leucocytozoon caulleryi. A transgenic potato clone with the highest rR7 antigen expression (3 µg rR7 antigen per gram of fresh-weight potato leaves) was selected, cultivated, and used in oral administration experiments to examine its ability to boost immunity. Chickens were immunized with chicken leucocytozoonosis vaccine “Hokken” by injection, and chickens that developed moderate levels of antibody titres were fed with TrP/R7 leaves. Chickens fed with TrP/R7 leaves showed increased antibody responses. In contrast, chickens fed with non-transgenic potato leaves showed a continuous decrease in antibody titres. Furthermore, chickens fed with TrP/R7 potato leaves showed strong resistance against experimental challenge with L. caulleryi infection. This study demonstrates the use of a plant-based oral vaccine to boost immunity against a protozoan disease.

Synchronous development of Eimeria tenella in chicken caeca and utility of laser microdissection for purification of single stage schizont RNA

Eimeria tenella is recognized worldwide as a significant pathogen in the poultry industry. However, a lack of methods for isolating developing schizonts has hindered the use of transcriptome analyses to discover novel and developmentally regulated genes. In the present study, we characterized the long-term successive development of E. tenella in infected chicken caeca and assessed the utility of laser microdissection (LMD) for the isolation of schizont RNA. Developmental stages, including those of the first, second, and third-generation schizonts and gametocytes, were synchronous. Using LMD, only the mature second-generation schizonts were successfully excised from the lamina propria, and non-degraded RNA was purified from the schizonts. E. tenella-specific genes were amplified by reverse transcription polymerase chain reaction (RT-PCR). These results augment our understanding of the E. tenella life cycle, and reveal LMD as a potentially useful tool for gene expression analyses of the intracellular stages of E. tenella.

Anticoccidial effect of halofuginone hydrobromide against Eimeria tenella with associated histology

Halofuginone (stenorol) has been used as an effective anticoccidial reagent for decades but very little is known about its mode of action. In this study, chickens were inoculated with Eimeria tenella oocysts on 14-day-old and medicated with halofuginone at days 0, 1, 2, 3, 4, 5 and 6 post inoculations (groups 0, 1, 2, 3, 4, 5 and 6, respectively). Chickens in group 7 were taken as challenge-unmedicated control and in group 8 unchallenged-unmedicated control. The survival rate, body weight gains (BWG), oocysts production, cecal scores, bloody diarrhea and histological examinations were analyzed to evaluate the anticoccidial efficacy of halofuginone and to initially elucidate its mechanisms. Results showed that halofuginone which acted as a coccidiostatic can significantly enhance the BWG, and decrease both the oocyst shedding and cecal destruction caused by E. tenella infection. The histological slide examination noted that halofuginone was effective when provided 0-2 days post inoculation but only partially effective when applied 3-7 days post infection. The second-generation schizonts treated with halofuginone appeared vacuolated and degenerated. It is concluded that halofuginone can inhibit the parasite's invasion of host cecal hypothetical cell at the early stages of life cycle and later disturb the parasite's development by vacuolation of the schizonts. The resulting abnormal schizonts could not divide into schizoites and were eventually eliminated by the host's immune response.

A rapid assay for detecting antibody against leucocytozoonosis in chickens with a latex agglutination test using recombinant R7 antigen

A method for detecting antibody against leucocytozoonosis in chickens by latex agglutination (LA) was developed using latex beads coated with recombinant R7 (rR7), an outer membrane antigen of second-generation schizonts of Leucocytozoon caulleryi. Compared with the agar gel precipitation test, which is widely used in Japan, LA could detect antibody induced by L. caulleryi infection with greater sensitivity. No agglutination was detected with sera from specific pathogen free or layer chickens that had not been vaccinated with oil-adjuvanted rR7 antigen, or infected with L. caulleryi, nor with sera from chickens inoculated with other pathogens, establishing the specificity of the assay. The LA test was also able to be used to quantify serum antibody induced by vaccination, which is not possible with the agar gel precipitation test. This study has shown that LA using the rR7 antigen is a simple, quick, and useful method for detecting antibody against L. caulleryi.

Expression of anti-apoptotic factors in cells parasitized by second-generation schizonts of Eimeria tenella and Eimeria necatrix

Intracellular infections by parasites require a functional anti-apoptotic mechanism for parasite survival within the host cell. The intracellular cycle of Eimeria tenella and Eimeria necatrix in chicken intestinal cells involves the maturation of schizonts within the epithelial cells lining the crypt lumen of the ceca ( E. tenella) and jejunum ( E. necatrix). After invasion, these cells detach from the epithelial layer and migrate into the underlying connective tissue, where maturation of second-generation schizonts takes place. However, the detached epithelial cells that harbour the parasite and localize in the lamina propia do not undergo apoptosis despite the fact that they are parasitized cells and are located in an inappropriate microenvironment. In this study we consider the hypothesis that E. tenella and E. necatrix may inhibit the host cell apoptosis that accompanies parasite-mediated transformation during late schizogony. To that end, the expression of both NF-κB, a transcriptional factor that blocks parasite-induced apoptosis, and bcl-x L, an anti-apoptotic protein induced by NF-κB, were studied in the host cell during the maturation of second-generation schizonts. In addition, the expression of the phosphorylated inhibitor of NF-κB, p-IkB α, was also studied to further confirm NF-κB activation. Immunocytochemical techniques, flow cytometric and blott analysis were applied by using polyclonal antibodies that specifically react with bcl-x L, p-IkB α, and NF-κB to detect these anti-apoptotic proteins in the parasitized cell. Our results offer evidence that both these coccidial species first induce NF-κB activation to protect the transformed parasitized cells from apoptosis, allowing the second-generation schizonts to mature, and later, after complete schizonts maturation, cause NF-κB inhibition to trigger host cell apoptosis in order to facilitate the escape of merozoites. To determine whether inhibition of the NF-κB pathway would induce apoptosis of the host cell, a protease inhibitor (TPCK), which induces apoptosis by mediating inhibition of IkB phosphorylation, was administered to parasitized chickens.

Cellular immune responses in chickens induced by recombinant R7 Leucocytozoon caulleryi vaccine.

We have recently developed recombinant subunit vaccine consisting of second-generation schizont (2GS) membrane protein (rR7) of Leucocytozoon caulleryi. Chickens immunized with rR7 antigen acquired clear resistance to challenge by Leucocytozoon sporozoites. We examined the induction of cellular immune responses in vaccinated chickens. Spleen adherent cells from vaccinated chickens showed significantly higher phagocytic activity against 2GS-coated latex particles than did cells from adjuvant-inoculated or untreated control birds. Anti-R7 chicken IgG significantly increased the phagocytic rate of adherent cells from these 3 groups. These results show that specific cellular immune responses are induced by recombinant R7 subunit vaccine consisting of L. caulleryi 2GS protein, which suppresses the growth of parasites in the host in association with antiparasite antibodies to 2GS antigen.

Characterization of Monoclonal Antibodies against the Second-Generation Schizonts of Leucocytozoon caulleryi.

Monoclonal antibodies (MAbs), R1 and M5, were established against the second-generation schizont of Leucocytozoon caulleryi (L. caulleryi). Both antibodies reacted to membrane and internal structure proteins of the second-generation schizont by immunofluorescence microscopy. Molecular weight of the second-generation schizont (2GS) antigen was analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. At least 40 protein bands were detected in 2GS antigen by SDS-PAGE under reduced condition and ranged from 10 to 270 kDa. MAb R1 reacted to polypeptides of 150-268 kDa in 2GS antigen, whereas MAb M5 did with that of 66 kDa. Injection with a protein of 2GS antigen fractionated by affinity chromatography using MAbs R1 and M5 protected chickens against challenge with sporozoites of L. caulleryi. These results suggest that MAbs, R1 and M5, recognize 2GS antigen of L caulleryi.

The correlation of protective effects and antibody production in immunized chickens with recombinant R7 vaccine against Leucocytozoon caulleryi.

Protective effects of recombinant R7 (rR7) vaccine against Leucocytozoon caulleryi in chickens were studied. After injection of oil-adjuvanted rR7 vaccine into chickens, antibody titers against second-generation schizonts (2GS) antigen of L. caulleryi (anti-2GS antibody) rapidly rose in all the immunized chickens, reached to a peak value 2 weeks after injection, and the titers persisted through 4 or 5 months after injection. Chickens having high levels of the anti-2GS antibody titers (> or = 102,400 ) at pre-challenge completely protected against sporozoites challenge of L. caulleryi. After the challenge inoculation, relatively high parasitemia of L. caulleryi was observed in all the inadequately immunized chickens having low levels of the antibody titers (< or = 3,200) at pre-challenge, although some of them seemed to be clinically normal. Correlation of protective effects in the immunized chickens was observed between both prevention of appearance of clinical signs and parasitemia after parasites challenge and anti-2GS antibody titers of the chickens at pre-challenge. The present study shows that chicken leucocytozoonosis can be prevented by vaccination, and humoral immunity may play an important role in the control of chicken leucocytozoonosis.

Pathogenicity of Leucocytozoon caulleryi for specific pathogen-free laying hens.

The pathogenicity of Leucocytozoon caulleryi against specific-pathogen-free laying hens was investigated. Many large schizonts (second-generation schizonts) of L. caulleryi were seen in the ovary and oviducts of chickens. Edema and pressure atrophy of the adjacent tissues were associated with these schizonts. The eggshell-secreting portion of the uterus exhibited the most severe damage in the oviduct. This experiment reconfirms that L. caulleryi may stop egg production in laying hens, presumably as a result of damage to ovaries and oviducts.

Role of reticulocytes on gametocytogenesis in chickens infected with Leucocytozoon caulleryi.

Reticulocytes, known as late polychromatic erythrocytes, were induced in blood of chickens infected with Leucocytozoon caulleryi by bleeding when the second-generation merozoites were released into the blood from the second-generation schizonts. The second-generation merozoites preferentially invaded into reticulocytes and developed to stage II gametocytes. Enhanced development of stage II gametocytes to mature gametocytes was observed in the reticulocytes in vivo and in vitro in the bleeding group. Nevertheless, invasion of reticulocytes by the second-generation merozoites was not considered to be absolutely necessary for the development of gametocytes.

Immunohistochemical identification of the cells parasitized by second-generation schizonts of Eimeria tenella.

Conflicting reports exist in the literature concerning the type of cells within the lamina propria of the ceca that harbor second-generation schizonts of Eimeria tenella. Most of the previous studies concerning these cells have been performed using routine light or electron microscopy. Consequently, difficulties are evident in precise definition of the type of these cells using normal morphological criteria, since growth of the schizonts of E. tenella alters the morphology of the parasitized cell, making it difficult to recognize the cell type. This has led us to investigate the possibility of precisely identifying the subepithelial cells that are parasitized by mature schizonts. For this purpose we used cytoskeletal markers, namely, keratin and vimentin intermediate filaments, which allow the discrimination between epithelial and mesenchymal cells. Localization of keratin and vimentin on frozen cecal sections was studied immunohistochemically using specific monoclonal antibodies. Sites of antigenicity were detected by the avidin-biotin complex (ABC) immunoperoxidase technique and visualized by the deposition of diaminobenzidine. The identity of the cells was confirmed by the immunodetection of keratin intermediate filaments in the cytoplasm of the cells. Immunoreactivity for vimentin was absent in the parasitized cells. Therefore, we conclude that the development of second-generation schizonts of E. tenella takes place in epithelial cells within the lamina propria, which are presumably crypt epithelial cells that leave the crypts and enter the lamina propria after infection by first-generation merozoites.

In vitro cultivation of the vascular phase of Sarcocystis singaporensis.

To establish an in vitro culture system for the precystic phase of Sarcocystis singaporensis, we initially tested various excysting fluids for sporocysts. An excysting fluid containing 2.5% bovine taurocholate and 10% bile of the specific intermediate host, Rattus norvegicus, in RPMI medium was the most suitable resulting in excystation of 80% of the sporozoites. Subsequently, we identified brain endothelial cells and pneumonocytes of the rat to promote growth of sporozoites to schizonts. Hepatoma, fibroblastic, or myoblastic cells were not suitable for the parasite's development. First-generation schizonts were seen at days 3-10 postinoculation (PI); a distinct second peak of schizogonic development only occurred in endothelial cells at days 14-18 PI. First-generation schizonts were 26.0 (+/- 3.8) microns in diameter and contained 32-50 merozoites, second-generation schizonts measured 34.4 (+/- 10.6) microns and contained 54-72 merozoites. Merozoite yield at large-scale culture conditions (75 cm2 flasks) using pneumonocytes as host cells was relatively low. Ultrastructurally, sporozoites and merozoites were quite similar to corresponding stages of other Sarcocystis species. With regard to host cell specificity and developmental kinetics, in vitro cultivation showed close similarities to the situation in vivo.

Characteristics of the anticoccidial activity of 6-azauracil.

The characteristics of anticoccidial activities of 6-azauracil (AzU) were investigated in the battery trials utilizing the White Leghorn cockerels, which were infected with Eimeria tenella, E. necatrix, E. acervulina, E. maxima and/or E. brunetti. AzU was mixed into the basal starter feed and fed ad libitum to the birds from 1 day before the inoculation of oocysts to the time of necropsy. AzU showed remarkable anticoccidial activity against E. tenella and E. necatrix infections at doses of 1,000 ppm in feed or more, and fairly good effect against E. acervulina infection at the dose of 4,000 ppm in feed. Inadequate effect against E. maxima infection and null in effect against E. brunetti at the dose of 4,000 ppm were resulted for this drug. The effect of 6-azauridine, ribonucleoside of AzU against E. tenella and E. necatrix infections at 1,000 ppm was tested and revealed negative results. In the test with AzU-resistant line of E. tenella, cross resistance was confirmed between AzU and emimycin riboside, a uridine analogue. Activity of AzU was studied in vitro against E. tenella in chick kidney cells. AzU inhibited the development of the first- and second-generation schizonts at 100-200 ppm in the medium. Degenerated parasites were observed.

The Ultrastructural Effect of Amprolium Medication on Development of Eimeria adenoeides in Turkeys

The ultrastructural appearance of first-generation schizonts of Eimeria adenoeides was markedly altered in turkey poults fed 0.0125% amprolium-medicated feed. When compared with development seen in unmedicated control birds 48-72 hr postinoculation (PI), most of the schizonts present in the medicated birds were fragmented, contained enlarged nuclei, had swollen endoplasmic reticulum and Golgi apparatus, and showed no budding of merozoites. Other schizonts were almost completely degenerated and contained pyknotic nuclei, dense cytoplasm, and large intracytoplasmic vacuoles containing membrane whorls or lipid droplets. A few mature schizonts were seen in the medicated poults, and these did not appear to differ ultrastructurally from those seen in unmedicated control birds. Ultrastructurally normal second-generation schizonts and the sexual stages were also seen in small numbers in the medicated turkey poults 72-120 hr PI. Mature sexual stages were seen much earlier in medicated turkeys--at 96 hr PI--than in the unmedicated poults.

Some aspects of Leucocytozoon caulleryi reinfection in chickens.

The role of first- and second-generation schizonts in acquired immunity to Leucocytozoon caulleryi in chickens was studied. The chickens, which had recovered from a primary infection with various doses of sporozoites at 22-95 days of age, were challenged with sporozoites. First-generation merozoites were found in all of the challenged chickens, but no second-generation merozoites and gametocytes were seen in 30 of 32 chickens challenged with sporozoites. Almost all of the chickens that had recovered from a primary infection with sporozoites showed complete resistance to reinfection, and those that had recovered from a primary infection with first-generation merozoites showed resistance to reinfection with sporozoites. These results indicate that the second-generation schizont of L. caulleryi appears to be more immunogenic than the first-generation schizont and that some immune factors acquired by the chickens in the second generation of schizogony may inhibit the development of second-generation schizonts.

Eimeria tenella: Use of a monoclonal antibody in determining the intracellular fate of the refractile body organelles and the effect on in vitro development

A monoclonal antibody, which recognizes the refractile body of Eimeria sporozoites, was used to study the developmental fate of this organelle during asexual development of E. tenella and to determine the effect of this monoclonal antibody on in vitro development of the parasite. Through use of immunofluorescent antibody and gold-labeling techniques at the light and electron microscopy level, the refractile body at 48 to 96 hr postinoculation was found to separate into 6 to 10 small globules, then diffuse throughout the schizont cytoplasm, and eventually reconcentrate as a small dot of material in each of the mature first-generation merozoites. The schizont did not develop to maturity if diffusion of the refractile body did not occur. The refractile body material was quickly lost as the merozoite left the schizont and invaded new cells and was not detected in any later developmental stages. The in vitro development of first- and second-generation schizonts of E. tenella was greatly inhibited (up to 100%) with exposure to the monoclonal antibody. There was an increase in the number of schizonts with nondispersed refractile body in the monoclonal antibody-treated cells when compared to the untreated controls, and the few mature schizonts seen had up to a 50-fold decrease in the number of merozoites. Immunofluorescent antibody labeling of the refractile body of intracellular sporozoites and schizonts treated in vitro with the monoclonal antibody for 24–96 hr postinoculation indicated that the antibody had crossed the host cell and parasite plasma membrane during incubation.

In vivo Action of the Anticoccidial Diclazuril (Clinacox[registered]) on the Developmental Stages of Eimeria tenella: An Ultrastructural Evaluation

A single 5-mg/kg oral dose of diclazuril affected both the asexual and sexual development of Eimeria tenella in experimentally inoculated chickens. In second-generation schizonts, early growth and nuclear divisions progressed normally, but a marked inhibition of merozoite formation was observed. Exogenesis of merozoites was largely prevented, whereas production of micronemes, amylopectin granules, and dense bodies and the formation of rhoptries, conoid, and pellicle continued. All these subcellular organelles accumulated, together with differentiated nuclei, within the main cytoplasmic mass. In the end, complete necrosis of the schizonts occurred. In macrogamonts, dilation of the rough endoplasmic reticulum around type II wall-forming bodies, fusion of type II wall-forming body contents, disturbance of the normal parallel arrangement of rough endoplasmic reticulum, and disruption of row formation of amylopectin granules became evident. In the microgamonts, normal evagination of microgametes was prevented; the flagellar complex formed within the main cytoplasmic mass and the differentiated nuclei remained present within the parasite body. The macro- and microgamonts also ended up in a stage of complete necrosis. These data indicate that diclazuril treatment primarily affects the normal differentiation of the respective endogenous stages during parasite development. This leads to complete degeneration of schizonts and gamonts indicating the lethal effect of this new anticoccidial compound.