The effect of dietary protein level on performance characteristics of coccidiosis vaccinated and nonvaccinated broilers following mixed-species Eimeria challenge.

A series of experiments were conducted to investigate the effect of starter diet protein levels on the performance of broilers vaccinated with a commercially available live oocyst coccidiosis vaccine before subsequent challenge with a mixed-species Eimeria challenge. Data indicated that an increasing protein concentration in the starter diet improved broiler performance during coccidiosis vaccination. Prechallenge performance data indicated that vaccination could decrease BW and increase feed conversion ratio. The time period most important for the observed effects appeared to be between 13 and 17 d of age. This reduction in performance parameters of vaccinated broilers compared with nonvaccinated broilers was eliminated by the conclusion of the experiments (27 d) in the diet groups with higher protein. Vaccination was effective at generating protective immunity against Eimeria challenge, as evidenced by increased (P < 0.05) BW gain, improved feed conversion, reduced postchallenge mortality, and reduced lesion development in vaccinated broilers compared with nonvaccinated broilers. These observations support numerous other reports that confirm live oocyst vaccination can be used effectively as a preventive against avian coccidiosis in commercially reared broilers. More important, these findings suggest that reduced protein concentration of starter diets can lead to significant losses in broiler performance when using a vaccination program to prevent coccidiosis.

Immunostimulating complexes incorporating Eimeria tenella antigens and plant saponins as effective delivery system for coccidia vaccine immunization.

Immunostimulating complexes (ISCOMs) are unique, multimolecular structures formed by encapsulating antigens, lipids, and triterpene saponins of plant origin, and are an effective delivery system for various kinds of antigens. The uses of ISCOMs formulated with saponins from plants collected in Kazakhstan, with antigens from the poultry coccidian parasite Eimeria tenella, were evaluated for their potential use in developing a vaccine for control of avian coccidiosis. Saponins isolated from the plants Aesculus hippocastanum and Glycyrrhiza glabra were partially purified by HPLC. The saponin fractions obtained from HPLC were evaluated for toxicity in chickens and chicken embryos. The HPLC saponin fractions with the least toxicity, compared to a commercial saponin Quil A, were used to assemble ISCOMs. When chicks were immunized with ISCOMs prepared with saponins from Kazakhstan plants and E. tenella antigens, and then challenged with E. tenella oocysts, significant protection was conveyed compared to immunization with antigen alone. The results of this study indicate that ISCOMs formulated with saponins isolated from plants indigenous to Kazakhstan are an effective antigen delivery system which may be successfully used, with low toxicity, for preparation of highly immunogenic coccidia vaccine.

cDNA encoding an immunogenic region of a 22 kilodalton surface protein of Eimeria acervulina sporozoites.

cDNA encoding an immunogenic region of a 22 kDa surface protein of Eimeria acervulina sporozoites was cloned and expressed in the bacteriophage lambda gt11 vector. The recombinant beta-galactosidase fusion protein, designated MA1, has an apparent molecular size of 125 kDa. Immunofluorescence staining of intact E. acervulina sporozoites and merozoites and immunoblotting of 125I-surface labeled protein from both stages revealed exclusive expression of the cloned cDNA in the sporozoite stage. The gene encoding the 22 kDa surface protein appears to exist as a single copy sequence as revealed by Southern blot hybridization utilizing the cDNA insert as a probe. Although not recognized by immune serum, purified recombinant MA1 antigen induced significant in vitro activation of T lymphocytes obtained from chickens immune to E. acervulina. DNA sequencing and hydropathic analysis of the predicted amino acid sequence revealed a central hydrophilic region surrounded by two hydrophobic areas which may represent exposed and transmembrane regions of the protein.

Use of live oocyst vaccines in the control of avian coccidiosis: experimental studies and field trials.

Areas addressed in this study on the use of live oocyst vaccines to control coccidiosis include: the influence of immunocompetency of the strains and sex of the birds used; methods of delivery of vaccine; immunological variation between different strains of the same coccidial species; and the effects of combining vaccine with anticoccidial medication. The results show that vaccination with live oocysts elicited significant protection against coccidiosis, both with experimentally induced and naturally acquired coccidial infection, resulting in average bird weight gains and feed efficiency similar to that obtained with conventional anticoccidial medication.

Dietary modulation of avian coccidiosis.

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.

Analysis of immunological cross-protection and sensitivities to anticoccidial drugs among five geographical and temporal strains of Eimeria maxima.

Two laboratory strains (USDA strain No. 68 isolated from the eastern shore of Maryland 15 years ago and a University of Guelph strain isolated from an Ontario broiler house 23 years ago) and 3 recent field strains of Eimeria maxima [isolated in Maryland (MD), North Carolina (NC) and Florida (FL)] were tested for their ability to induce cross-protective immunity and their sensitivities to a variety of anticoccidial compounds. To assess immunological cross-protection, 1-day-old chicks were inoculated and subsequently challenged at 10 days of age, testing all possible combinations of initial inoculating (immunizing) and subsequent challenge strain. Six days post-challenge, chicks were killed and weight gains and lesion scores were determined and compared to sham inoculated and challenged, and sham challenged age-matched controls. The 2 laboratory strains and the NC strain were fully cross-protective against each other by both these measures. In contrast, the MD and FL strains induced complete protection only against the homologous strain. Reciprocally, no other strains protected chicks completely against the FL and MD strains. Drug sensitivity studies using 10 different anticoccidial formulations at prescribed drug levels showed significant differences between the 2 laboratory strains and the 3 recently isolated field strains; more recent isolates from commercial broiler houses demonstrated complete or partial resistance to a wider range of anticoccidial compounds. No correlation was seen between cross-protection and sensitivities to anticoccidials.

Analysis of infraspecific variation among five strains of Eimeria maxima from North America.

Two laboratory strains from the eastern shore of Maryland 15 years ago and from an Ontario broiler house 23 years ago and three recent field strains of Eimeria maxima (isolated in Maryland, North Carolina and Florida) were examined for phenotypic and genotypic variation using protein profiles, random amplified polymorphic DNA-PCR analysis and DNA sequences obtained from the internal transcribed spacer regions of the rRNA genes. Staining profiles obtained by one-dimensional SDS-PAGE of sporozoite proteins were identical in all five strains. Using random amplified polymorphic DNA-PCR analysis with high %G-C content decamers as primers, we were able to confirm that the five strains are all E. maxima, but were unable to discern any relationships among them because of the limited number of shared polymorphisms identified. In contrast, cloning and sequencing of the internal transcribed spacer-1, 5.8S rDNA and internal transcribed spacer-2 regions of the rRNA genes provided sufficient sequence information to infer phylogenetic relationships among the strains. Almost all of the infraspecific variation was located in the internal transcribed spacer regions. Only two base changes were identified within the 5.8S rRNA gene. Evolutionary relationships among the strains inferred using parsimony analysis of the aligned internal transcribed spacer sequences were well supported, but the hypothesised relationships did not correlate well with the demonstrated immunological cross-reactivities of these strains.

Sustainable coccidiosis control in poultry production: the role of live vaccines.

The development of new methods of administering coccidiosis vaccines has facilitated their use in the hatchery and thereby improved prospects for the economic vaccination of broilers. The acquisition of protective immunity to Eimeria species is boosted by further exposure to infection after vaccination. Factors that affect the reproductive efficiency of non-attenuated and attenuated vaccines are considered and the key role that oocyst production plays in establishing and maintaining uniform immunity in a flock of chickens is discussed. In addition to immunisation, a possible advantage to the application of certain vaccines is that their use could repopulate poultry houses with drug-sensitive organisms. Theoretical rotation programmes in which the use of drugs is alternated with that of vaccines are described. Variability of the cross-protective immune response between strains of the same species should be considered during vaccine development and subsequent use. The significance of less common species of Eimeria, not included in all vaccines, also needs to be assessed. An important consideration is the occurrence of pathogens other than Eimeria (such as the bacterium Clostridium) in flocks given coccidiosis vaccines and the methods by which they might be controlled. More research is required into the relationship between bacterial and viral infections of poultry and coccidiosis vaccination. Vaccines need to be developed that are simple to apply and cost effective for use in areas of the world where small-scale poultry production is commonplace. In the near future it is likely that more live vaccines based upon oocysts derived from attenuated strains of Eimeria will be developed but in the longer term vaccines will be based on the selective presentation to the host of specific molecules that can induce protective immunity. This achievement will require significant investment from the private and public sectors, and, if successful, will facilitate the sustainable control of coccidiosis in poultry production.

Inhibition of Plasmodium berghei sporozoite invasion of cultured hepatoma cells.

Monoclonal antibody to a Plasmodium berghei surface antigen blocked sporozoite invasion of cultured human hepatoma cells. Such antibodies were of IgG1 class. Two monoclonals of the IgM class, probably reactive with the same antigen, did not neutralize invasion. It appears that the sporozoite surface antigen mediates invasion of hepatoma cells.

Retention of Plasmodium berghei sporozoites within perfused mouse livers.

A mouse liver perfusion model was adapted to evaluate the efficiency of the liver in retaining Plasmodium berghei sporozoites. Specific numbers of sporozoites were perfused into each liver via a portal vein cannula. The numbers of sporozoites in the perfusate effluent were counted and the percent sporozoite retention calculated. Over 95% of sporozoites suspended in medium with plasma were retained in a normal liver following a single passage. Sporozoites were seen in sinusoids of perfused livers using scanning electron microscopy. This liver perfusion model offers a valuable method to help clarify sporozoite interactions with elements of the liver.

The effects of Eimeria acervulina infection on the metabolism of chick duodenal tissue.

Broiler chicks, 2--3 weeks old were infected with eimeria acervulina, and the metabolism and ultrastructure of the infected duodenal tissue were studied during the period 3--14 days after inoculation (DAI). Between 4 and 5 DAI duodenal rings showed an increase in C-1/C-6 ratios of CO2 evolved from glucose as well as decreases in the rates of oxidation of glucose and octanoic acid. Between 4--7 DAI mitochondria from infected epithelial layers had reduced rates of octanoic acid and alpha-ketoglutaric acid oxidation as compared to controls. Electron microscopic observations confirmed the biochemical findings. At 5--6 DAI mitochondria in many uninfected cells were progressively swollen and then vacuolated as the cristae appeared to break down. Mitochondria in cells which contained parasites did not show these changes.

Specificity and cross-reactivity of hybridoma antibodies generated against Eimeria bovis sporozoites.

Spleens from mice immunized with Eimeria bovis sporozoites were removed and the cells fused with mouse myeloma cells to produce hybridoma cell lines (HCLs). The resulting HCLs were examined for antibody (HAB) production against E. bovis sporozoites using an indirect immunofluorescent antibody test on air-dried sporozoites. Four fusions resulted in the production of 19 HCLs that produced HABs to E. bovis sporozoites. These 19 HCLs were further tested for reactivity with cell culture-grown merozoites of E. bovis and Sarcocystis cruzi of cattle; sporozoites of Eimeria tenella from chickens, Eimeria meleagrimitis from turkeys, Eimeria papillata and Eimeria vermiformis from mice; and bradyzoites of S. cruzi from calves. Six HCLs produced HABs that reacted only with E. bovis sporozoites and were species specific/stage specific. Two HCLs produced HABs that reacted only with E. bovis sporozoites and merozoites, and were species specific/stage cross-reactive. Seven HCLs produced HABs that reacted with the sporozoites of the other Eimeria species examined and were species cross-reactive/stage specific. Four of the HCLs produced HABs that reacted with all organisms tested and were species cross-reactive/stage cross-reactive. The results of this study suggest the conservation of some antigens throughout developmental stages and genera of Eimeriorina.

Development and use of hybridoma antibodies directed against Eimeria acervulina merozoites for cross-reactive and ferritin-labeling studies.

Hybridoma antibodies (Hab) were produced against Eimeria acervulina merozoites that had been separated from extraneous intestinal material by a fiber column technique before injection into mice. The Hab demonstrated three different immunofluorescent-antibody (IFA) patterns of tip, surface, or surface-internal fluorescence in or on the merozoites. Some Hab reacted with round immature schizonts, which were also present in the fiber-cleaned merozoite material. Variations in cross-reactivity were seen with a number of Hab tested by IFA with merozoites, sporozoites, and immature schizonts of different coccidial species. Certain Hab were species- and stage-specific, whereas others cross-reacted with some or all stages or species tested. One Hab apparently reacted with only a small percentage of the E. acervulina merozoites in the fiber-cleaned material. The ferritin (Fe)-labeling technique showed that with one Hab, which gave a surface-internal IFA pattern, there was an irregular clumping of the Fe label along the surface of the immature schizont. A heavier deposit of Fe label was seen on the area of the schizont where the merozoite was beginning to form. A heavy uniform labeling of Fe was seen on the surface of the pellicle of the mature merozoites. These results demonstrate that stage-specific and cross-reactive antigens are present in or on the merozoites of E. acervulina, and as shown with one Hab, surface antigens present on the immature schizont are incorporated onto the mature merozoite.

Use of hybridoma antibodies and recombinant DNA technology in protozoan vaccine development.

The use of hybridoma antibodies developed against the sporozoite stage of avian coccidia, coupled with genetic-engineering techniques, has made it possible to begin bird-immunization studies utilizing an Escherichia coli-elicited coccidial protein. The coccidia are currently controlled in the poultry industry by use of anticoccidial compounds, but it now may be possible to use the bird's own immune system for defense against the parasitic infection. Since the sporozoite stage, which initiates the infection in poultry, is quite complex and is made up of hundreds of proteins or antigens, hybridoma antibodies were produced to identify specific antigens. These antigens, once identified, were found in such minute amounts that it became necessary to utilize genetic engineering in order to produce enough protein for immunization studies. One such protein, designated 5401, has been shown to stimulate an antibody response in immunized birds and to impart partial protection against a coccidial challenge infection. The results of these studies indicate that development of a vaccine against coccidial parasites may someday be possible.

A study of the dynamics of the invasion of immunized birds by Eimeria sporozoites.

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.

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.

Eimeria tenella and E. acervulina: differences in ability to elicit cross-species protection as compared with the turkey coccidium, E. adenoeides.

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.

Influence of betaine and salinomycin on intestinal absorption of methionine and glucose and on the ultrastructure of intestinal cells and parasite developmental stages in chicks infected with Eimeria acervulina.

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.

Avian eimeria: invasion in foreign host birds and generation of partial immunity against coccidiosis.

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.

Effects of hybridoma antibodies on invasion of cultured cells by sporozoites of Eimeria.

Hybridoma antibodies (Hab) produced against sporozoites or merozoites of four species of Eimeria were tested for the ability to inhibit the invasion of cultured primary avian kidney cells by sporozoites of Eimeria. Five of 16 Hab that were tested showed inhibitory activity. All five of these Hab were produced against sporozoites and reacted with sporozoite surface antigens or surface/internal antigens. Four Hab produced against merozoites of E. acervulina cross-reacted with sporozoite surface antigens but failed to inhibit invasion. Similarly, Hab reacting with sporozoite anterior tips or refractile bodies had little effect on invasion. Collectively, the data suggest that surface antigens or surface/internal antigens that are unique to the sporozoite stage may influence or be part of the invasion process. Indirect immunofluorescent-antibody tests and ferritin (Fe) labeling combined with electron microscopy indicated differences in binding of two of the Hab to the sporozoite surface membranes. For example, after exposure to Hab 43A6 and a fluorescein-antimouse IgG conjugate, extracellular sporozoites of E. meleagrimitis fluoresced brightly but intracellular sporozoites exhibited little fluorescent label. Sporozoites labeled with Hab 43A6 plus a ferritin-antimouse IgG conjugate that were observed in the process of cell invasion had ferritin on the extracellular portion of the parasite but not on the intracellular portion. Extracellular aggregates of ferritin were observed near the site of invasion. The data suggested that antigens of the sporozoite surface that are recognized by Hab 43A6 are "scraped off" during the invasion of cells. In contrast, after exposure to Hab E5, both extracellular and intracellular sporozoites of E. tenella fluoresced. However, ferritin label was not observed on viable sporozoites, even when they were fixed immediately after the labeling procedure. The antigens recognized by Hab E5 may be associated with parasite secretory products rather than with an integral part of the sporozoite surface membrane.