Enhanced egress of intracellular Eimeria tenella sporozoites by splenic lymphocytes from coccidian-infected chickens

Egress, which describes the mechanism that some intracellular parasites use to exit from parasitophorous vacuoles and host cells, plays a very important role in the parasite life cycle and is central to Eimeria propagation and pathogenesis. Despite the importance of egress in the intracellular parasite's life cycle, very little information is known on this process compared to other steps, e.g., invasion. The present study was conducted to investigate the interplay between the host adaptive immune system and Eimeria egression. Splenic lymphocytes or soluble immune factors were incubated with parasite-infected host cells for 3 or 5 h, and the percentage of egress was calculated according to an established formula. Viability of egressed parasites and host cells was tested using trypan blue exclusion and annexin V and propidium iodide staining, respectively. We found that premature egression of sporozoites from Eimeria tenella-infected primary chicken kidney cells or from chicken peripheral blood mononuclear cells occurred when the cells were cocultured in vitro with spleen lymphocytes from E. tenella-infected chickens but not when they were cocultured with splenocytes from uninfected chickens. Eimeria-specific antibodies and cytokines (gamma interferon [IFN-γ], interleukin-2 [IL-2], and IL-15), derived from E. tenella-primed B and T lymphocytes, respectively, were capable of promoting premature egress of sporozoites from infected host cells. Both egressed parasites and host cells were viable, although the latter showed reduced reinvasion ability. These results suggest a novel, immune-mediated mechanism that the host exploits to interrupt the normal Eimeria life cycle in vivo and thereby block the release of mature parasites into the environment.     

Common epitopes on Eimeria tenella sporozoites and cecal epithelium of chickens.

Eimeria tenella sporozoites invade the intestinal epithelium at a highly specific site. Certain molecules that were recognized by monoclonal antibody E.TEN 11M-2, which was prepared against E. tenella sporozoites, were detected on both cecal epithelium and sporozoites. We expect that these molecules are involved in attracting sporozoites towards the site of entry.     

Eimeria adenoeides sporozoites: enhanced invasion of cultured cells previously inoculated with Eimeria acervulina sporozoites

The effects of prior or concurrent administration of Eimeria acervulina on invasion of cultured cells by Eimeria adenoeides sporozoites and possible mechanisms of action were examined. Baby hamster kidney (BHK) cell cultures that were inoculated with E. acervulina sporozoites were significantly more permissive for invasion by E. adenoeides sporozoites than uninoculated cultures. Enhancement of invasion by E. adenoeides did not occur when the two species were inoculated into cultures concurrently, or within 30 h of each other. However, 48 and 72 h after inoculation of BHK cells with E. acervulina, invasion by E. adenoeides sporozoites was significantly greater than invasion in uninoculated cultures. At 96 h postinoculation with E. acervulina, the enhancing effect on invasion was variable. Culture media collected from E. acervulina-inoculated cultures also significantly enhanced invasion by E. adenoeides. Slight changes in proteins of E. acervulina-inoculated versus uninoculated cell cultures were detected by Western blots of biotinylated and nonbiotinylated cells. Biotinylated bands between 10 and 25 kDa increased in the inoculated cultures. In addition, when chicken anti-E. acervulina sporozoite serum was used as a probe, labeling of a 10 kDa antigen increased in the inoculated cultures.     

Lectin-binding by sporozoites of Eimeria tenella

Sporozoites of Eimeria tenella were reacted in vitro with 19 different lectins characterized with a variety of carbohydrate-binding properties. Nine lectins caused sporozoite agglutination, which was inhibited by the specific carbohydrates mannose, sialic acid, melibiose, D-galactose, or D-galNAc. When intact live or fixed whole sporozoites were reacted with fluorescein isothiocyanate-conjugated lectins, another nine lectins bound to sporozoites, giving weak to strong fluorescence but not agglutination. Of these, all nine lectins bound to surface sites, but four also bound to the refractile body. Two of the agglutinating lectins also bound to intracellular organelles of air-dried sporozoites. SDS-PAGE analysis showed that biotinylated lectins bound a wide variety of parasite proteins. Lectins with similar carbohydrate specificities had some similarity in binding patterns of parasite proteins, as well as marked differences. In a few cases lectins with different carbohydrate specificities bound common protein bands. Only one lectin (Dolichos biflorus) showed no evidence of binding to whole sporozoites, organelles, or proteins.     

Studies on the viability of sporozoites of Eimeria tenella

Summary Sporozoites, obtained from oocyst cultures stored at 4° C for more than 65 days were markedly less viable than those hatched from new oocyst cultures. There appeared to be no quantitative differences in the viability of sporozoites stored in liquid N₂ for 99 days. Counts of macroscopic focal lesions on the chorioallantois of embryos infected with E. tenella appears to be an accurate method for assessing the viability of sporozoites of this species.     

Eimeria separata: method for the excystation of sporozoites

A method is described for the excystation and collection of infective sporozoites of Eimeria separata. The procedure uses conditions that resemble the in vivo environment. The first treatment of the oocysts in a 0.4% pepsin/HCl solution alters the oocyst wall, which becomes thinner. The second treatment in a 0.4% trypsin/0.75% taurocholate solution breaks the oocyst wall and sporocysts are released. A third incubation of the oocyst-sporocyst mixture in trypsin-free medium with 0.75% taurocholate and an additive of MgCl₂ followed by a final incubation in RPMI medium supplemented with 1% fetal calf serum yields a sporozoite excystation rate of up to 90%. Received: 23 October 1998 / Accepted: 17 November 1998     

Extraintestinal sporozoites of chickenEimeria in chickens and turkeys

Oocysts were found in the feces of chickens (recipients) dosed orally with whole blood, liver, lung, or heart homogenates from chickens and turkeys (donors) inoculated 3 and 4 days previously with a mixture of 3.5×10⁶ oocysts of chickenEimeria. No oocysts were found in the feces of recipients given spleen homogenates from these same chickens and turkeys and none were found in the feces of recipients given similar material from uninoculated donors. Intracellular sporozoites were found in the peripheral blood of a turkey inoculated with chickenEimeria. The results indicate that a small number of sporozoites are capable of invading and surviving for at least 4 days in the peripheral blood of chickens and turkeys.Supported by State and Hatch funds allocated to the Georgia Agricultural Experiment Stations of the University of Georgia     

Identification of immunodominant surface antigens of Eimeria acervulina sporozoites and merozoites

Immunodominant surface antigens of Eimeria acervulina sporozoites and merozoites were identified by 125I-labeling and immunoblotting studies. Using these methodologies 60% of the immunodominant sporozoite antigens and 90% of the immunodominant merozoite antigens were observed to be 125I-surface labeled. However, several major 125I-labeled sporozoite and merozoite proteins did not represent prominent antigens as measured by immunoblotting. Immunodominant surface antigens were found over a wide size range for sporozoites (21-110 kDa) and for merozoites (20-250 kDa). In order to relate these findings to a 'natural' infection, two groups of 3-week old chickens were inoculated 5 times over a 2.5 week period with either a low or high dose of E. acervulina oocysts. The serum response to sporozoites and merozoites, indicated by enzyme-linked immunosorbent assay titers, was rapid; less than or equal to 7 days post-infection with 10(4) oocysts and less than or equal to 3 days with 10(5) oocysts. Many of the antigens identified by immunoblotting of sera from sporozoite- and merozoite-immunized animals were recognized by sera from both high dose and low dose E. acervulina-infected chickens. Furthermore, the sporozoite and merozoite antigens could be grouped into those constituents which induced a serum response early or late in the infection.     

Purine metabolism in the intact sporozoites and merozoites of Eimeria tenella

Intact Eimeria tenella sporozoites and merozoites did not incorporate radiolabeled formate or glycine into their purine nucleotides suggesting a lack of de novo purine synthesis. However, [U-14C]glucose was incorporated into the cellular purine and pyrimidine nucleotide pools of both forms probably via conversion to radiolabeled ribose-1-phosphate and/or 5'-phosphoribosyl-1-alpha-pyrophosphate and the resulting action of various purine and pyrimidine salvage enzymes. Both forms of the parasite salvaged radiolabeled purine bases and nucleosides in a similar fashion. These purines were incorporated into ribonucleotides and into RNA and DNA. Adenine and inosine were transformed to hypoxanthine. Adenosine was converted to both inosine and hypoxanthine. Hypoxanthine and xanthine were not oxidized to uric acid but were metabolized to nucleotides. Guanosine was cleaved to guanine; guanine was deaminated to xanthine. The results demonstrate the presence of several purine salvage pathways. Purine phosphoribosylating and nucleoside phosphorylating activities as well as purine nucleoside cleaving and adenosine, adenine and guanine deaminating activities were evident. The metabolic evidence suggests the enzymes required to convert the newly formed nucleoside monophosphates to ATP and GTP were present also.     

Involvement of CD 8+ and CD 3+ lymphocytes in the transport of Eimeria necatrix sporozoites within the intestinal mucosa of chickens

The phenotype of cells transporting sporozoites of Eimeria necatrix during a primary infection was determined using a panel of six monoclonal antibodies to various chicken lymphocyte surface markers. Sporozoites and cells harboring them were examined at 8, 12 and 18 h postinfection using two-color immunoflorescence and confocal microscopy. The majority of parasites observed within lymphocytes were found in CD 8 + (15%) or CD 3+ (13-22%) cells at all time periods examined. Smaller numbers were found within deltagamma TCR+ (5%) and alphabeta TCR+ (5%) lymphocytes. No sporozoites were found within CD 4+ or IgM+ lymphocytes at any of the time periods.     

Invasion of the intestinal tract by sporozoites of Eimeria coecicola and Eimeria intestinalis in naive and immune rabbits

Naive and immune specific-pathogen-free rabbits were inoculated in the duodenum with sporocysts of Eimeria coecicola or Eimeria intestinalis. Samples were taken from the following tissues: duodenum (site of penetration of sporozoites), ileum (specific target site of the endogenous development of E. intestinalis), vermiform appendix (target site of E. coecicola) and two extraintestinal sites, mesenteric lymph nodes (MLNs), and spleen. The presence of sporozoites was checked by immunohistochemistry. In rabbits primary-infected with E. coecicola, large numbers of sporozoites were detected in the duodenum, extraintestinal sites, and vermiform appendix. The abundance of sporozoites in the spleen, MLN, and appendix was significantly reduced in the immune rabbits, and the migration seemed impeded. In the rabbits infected with E. intestinalis, sporozoites were absent in the spleen and MLN, indicating that the route of migration is different from that of E. coecicola. The number of sporozoites in the crypts of the ileum was markedly reduced in the immune animals.     

Effects of curcumin (diferuloylmethane) on Eimeria tenella sporozoites in vitro

The negative effects of coccidiosis on poultry health and productivity and increasing problems related to drug resistance have stimulated the search for novel and alternative methods of control. The present study evaluates the anticoccidial activity of curcumin (diferuloylmethane), a natural polyphenolic compound abundant in the rhizome of the perennial herb turmeric (Curcuma longa) which is a spice and food colourant commonly used in curries and also used as medicinal herb. Its effects were evaluated on Eimeria tenella sporozoites, including morphological alterations, sporozoite viability and infectivity to Madin–Darby bovine kidney (MDBK) cells. Morphological alterations of the sporozoites were recorded as deformation due to swelling and cell membrane corrugations. Curcumin at concentrations of 25, 50, 100, 200 and 400 μM showed considerable effects on sporozoite morphology and viability in a dose-dependent manner after incubation over 3, 6, 18 and 24 h while lower curcumin concentrations (6.25 and 12.5 μM) were not effective. In comparison to the untreated control, sporozoite infectivity was reduced at curcumin concentrations of 100 and 200 μM by 41.6% and 72.8%, respectively. Negative effects of curcumin on MDBK cells were not seen at these concentrations; however, curcumin at concentrations of 1,800, 600 and 400 μM was toxic to MDBK cells and affected cell proliferation. In conclusion, curcumin exhibited a marked inhibitory effect in vitro on E. tenella sporozoites inducing morphological changes and reducing sporozoite viability and infectivity.     

Shedding of the immunodominant P20 surface antigen of Eimeria bovis sporozoites.

P20 is an immunodominant surface antigen of Eimeria bovis sporozoites. As parasites underwent merogony within cultured bovine monocytes and Madin-Darby bovine kidney (MDBK) cells, P20 appeared to be shed gradually by meronts and was absent in type 1 and 2 first-generation merozoites. Meronts of E. bovis appeared to shed P20 into the parasitophorous vacuole of bovine monocytes, whereas MDBK cells evidently released P20 into the culture medium or destroyed its antigenic determinant.     

Determination of the purity of Eimeria species of chickens

The morphologic criteria length and breadth were used for the examination of the purity from 23 strains of different Eimeria species in the fowl (Eimeria tenella, E. acervulina, E. maxima, E. necatrix) by three methods: Tabulate graphic method, determination of the size of concentration, analysis with counter. The tabulate graphic method by use of peak value as only criterion of valuation has suitable proved for the screening test. A qualification of the degree of purity is reached in the form of classes of homogeneity with the criteria peak value, first and second classes of the neighbour by the determination of the size of concentration. The quantitative certainly of the assertion increases by the analysis with counter from six statistical parameters (frequency of peak value, frequency of the sum from peak value, first and second classes of the neighbour, number of taking classes in the specific range of species, coefficient of variation, coefficient of correlation, divergence of specific index of the form in the species).     

Characterization of a surface antigen of Eimeria tenella sporozoites and synthesis from a cloned cDNA in Escherichia coli

An antigenic surface protein of Eimeria tenella sporozoites has been identified that is the target of two neutralizing monoclonal antibodies Ptn 7.2A4/4 and Ptn 9.9D12. The antigen as isolated from the parasite is composed of a 17 kDa polypeptide and a 8 kDa polypeptide linked by a disulfide bridge. De novo synthesis of the antigen does not begin until approximately 16-20 h after the initiation of oocyst sporulation. A cDNA library was constructed using mRNA from sporulated oocysts and a clone encoding the antigen was isolated. The Ta4 gene encodes a single polypeptide of 25 kDa which contains the 17 and 8 kDa polypeptides. The protein has been synthesized in Escherichia coli either directly or as part of a beta-galactosidase fusion protein. The products synthesized in E. coli are single polypeptides and are not cleaved to two polypeptides as is seen in the parasite. The products accumulate in bacteria in an insoluble form which can be solubilized and renatured to an immunoreactive form.     

Monoclonal antibodies produced against sporozoites of the human parasite Plasmodium malariae abolish infectivity of sporozoites of the simian parasite Plasmodium brasilianum.

We have used a sporozoite neutralization assay to define the biological relevance of the cross-reactivity of two monoclonal antibodies, raised against sporozoites of the human parasite Plasmodium malariae (Uganda 1/CDC), with sporozoites of the simian parasite Plasmodium brasilianum (Colombian). In vitro incubation of each of these two monoclonal antibodies with sporozoites of P. brasilianum totally abolished the infectivity of these parasites for Saimiri sciureus. Using Western blot analysis and one of the P. malariae monoclonal antibodies, we identified two sporozoite proteins characteristic of the Colombian isolate of P. brasilianum with apparent molecular weights of 56,000 and 66,000. The same monoclonal antibody identified two proteins in an extract of the Peruvian isolate of P. brasilianum with apparent molecular weights of 59,000 and 69,000.     

Eimeria: immunisation of young chickens kept in litter pens

Infection of chickens with an attenuated strain of Eimeria tenella gave good protection against challenge with the parent strain of this species. Chickens given an attenuated strain of E. acervulina var. mivati were protected against challenge with other pathogenic cultures of E. acervulina when kept in litter pens under conditions allowing reinfection. Two-week-old chickens, kept in litter pens, were given attenuated strains of E. tenella and E. acervulina and small numbers of oocysts (50-100) of E. brunetti, E. maxima and E. necatrix. Body weight gain was unaffected and resistance to challenge infection was demonstrated at 6 and 9 weeks after inoculation. In other experiments only a small proportion of 1 to 3-week-old chickens in each pen were given oocysts. Chickens placed in contact with these 'seeder' chickens were immunised against five species of Eimeria within a 2 to 4 week period. Similar experiments with 1-week-old chickens showed that these could also be immunised but the degree of parasitism during immunisation caused some weight gain depression. These results suggest that controlled immunisation against several species of Eimeria may be possible and further experiments are needed under conditions which more closely resemble those operating in the field.