Biomedical subjects

H D Danforth

Publications and source records attributed to H D Danforth.

At least 37 records · Page 2Linked to original sources

PubMed1996

Diets high in n-3 fatty acids reduce cecal lesion scores in chickens infected with Eimeria tenella.

Four experiments were run in which diets incorporating various levels of n-3 fatty acids (n-3FA) from fish (menhaden) oil or flax seed were tested for their abilities to reduce the severity of Eimeria tenella infections in young broiler chicks. The diets were fed from 1 d of age through 3 wk of age. Chickens were infected at 2 wk of age. Diets consisting of broiler starter ration supplemented with 2.5 to 10% fish oil, 10% flax seed oil, or 10% linseed oil significantly decreased cecal lesions and maintained weight gains as compared to unsupplemented diets. Light micrographs of ceca from chickens that consumed high n-3FA diets showed reduced parasite invasion and development. Short-term feeding of diets high in n-3FA to young chicks may be a practical method for reducing production losses from cecal coccidiosis.

Animals↗

PubMed1996

Association of lowered plasma carotenoids with protection against cecal coccidiosis by diets high in n-3 fatty acids.

A series of four experiments was run to assess the effectiveness of diets containing high amounts of n-3 fatty acids in reducing the pathological effects of cecal coccidiosis in chickens caused by Eimeria tenella. To determine whether the dietary effects were related to development of oxidative stress, plasma samples were analyzed for tocopherols and carotenoids. Plasma vitamin E (alpha-tocopherol) values were not consistent between experiments. Total plasma carotenoids, however, were significantly decreased by 2.5 to 20% diet supplementation with fish oil in several experiments. These decreases coincided with significant reductions in lesion scores. Under the experimental conditions, total plasma carotenoid concentrations may serve as a sensitive indicator for oxidative stress, which may be a factor in reducing cecal lesions in E. tenella infections.

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PubMed1995

Gamma-irradiated and nonirradiated Eimeria tenella sporozoites exhibit differential uracil uptake and expression of a 7- to 10-kDa metabolic antigen.

Eimeria tenella sporozoites were exposed in the oocyst form to either an optimum (15 kRad) or a high (25 kRad) dose of gamma irradiation and used to infect cultured chicken embryo fibroblasts (CEF). The sporozoite-infected CEF monolayer was pulsed at time of infection or 24 hr postinfection with [3H]uracil and harvested 24 hr later to measure sporozoite metabolic activity. Sporozoites exposed to either 0 or 15 kRad gamma irradiation incorporated similar (P > 0.05) amounts of [3H]uracil during the first and second 24-hr periods after infection. However, there was a significant decrease (P < 0.05) in [3H]uracil uptake by 25 kRad-exposed sporozoites compared to nonirradiated and 15 kRad-irradiated sporozoites. Indirect immunofluorescence (IFA) staining of E. tenella sporozoite-infected CEFs using monoclonal antibodies (MAb) specific for somatic or "metabolic" antigens showed that gamma irradiation also affected the release of intracellular metabolites. Regardless of irradiation dose, extracellular sporozoites exhibited similar intensity of immunofluorescence when stained with either somatic antigen- or metabolic antigen-reactive MAb. Also, somatic antigen expression was similar for intracellular parasites irrespective of radiation dose. However, metabolic 7- to 10-kDa antigen expression by 25 kRad-irradiated sporozoites was markedly reduced compared to nonirradiated or 15 kRad-irradiated intracellular sporozoites. These results were corroborated by immunostaining sporozoite/CEF protein-impregnated Immobilon membrane with somatic or metabolic 7- to 10-kDa antigen-reactive MAb. These findings may indicate that the metabolic 7- to 10-kDa antigen is involved in protective immunity elicited by nonirradiated and/or 15 kRad-irradiated E. tenella sporozoites.

Animals↗

PubMed1994

Ultrastructural observations of development of Eimeria tenella in a novel established avian-derived cell line.

Transmission electron microscopy was used to study the in vitro development of Eimeria tenella in a novel established avian-derived cell line (designated CEV-1/F7) used for antigen production in chicken immunization studies. Sporozoites of E. tenella were inoculated onto cell monolayers and the cells were fixed at 24-h intervals. Large numbers of intracellular sporozoites were seen at 24 h postinoculation (p.i.), and trophozoites were identified at 24-48 h p.i. Immature schizonts, some with budding merozoites, were seen by 48 h p.i. At 72-96 h p.i., immature and mature schizonts and extracellular merozoites were observed. No merozoite invasion occurred, but immature second-generation schizogony was seen in parasitophorous vacuoles of first-generation schizonts. No further development occurred and degeneration of most schizonts was seen by 120-144 h p.i. The results confirmed synchronous development of E. tenella until 48 h p.i., followed by asynchronous development and ultrastructural degeneration with increased incubation time.

Animals↗

PubMed1994

In vitro and in vivo immunolabeling of sporozoites, schizonts, and sexual stages of Eimeria acervulina and E. tenella by a species- and stage-cross-reactive monoclonal antibody.

A cross-reactive monoclonal antibody (mAb), designated 1205, was used to study redistribution, parasitophorous vacuole (PV) incorporation, and in situ antigen production during the intracellular parasite development of Eimeria acervulina and E. tenella. Western-blot analysis of sporozoite preparations showed that the mAb recognized antigenic bands at 55 and 80 kDa. Indirect immunofluorescent antibody (IFA) labeling of sporozoites produced an internal dot pattern. Immunogold electron microscopy (IM) showed labeling of dense granules within sporozoites. The IFA pattern changed to a general-internal label in immature schizonts followed by a surface-tip pattern in mature merozoites both in vitro and in vivo. IM of the asexual stages revealed the same labeling pattern for the in vivo development of both species, and labeling of rhoptries was seen. In vitro, the PV membrane together with amorphous material within the PV was labeled by IFA during schizont development for E. tenella. No IM labeling of either the PV membrane or material within the PV was observed. Sexual stages seen in vivo for both species had the general-internal IFA pattern.

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PubMed1993

Ultrastructural observations of host-cell invasion by sporozoites of Eimeria papillata in vivo.

Scanning and transmission electron microscopy were used to study the invasion of mouse small-intestinal epithelium by sporozoites of Eimeria papillata. Some mice received oocysts by gavage and others received either sporocysts or sporozoites by direct injection into the small intestine. The highest concentration of invaded cells were found in ligated intestinal tissues studied at 5-45 min after the inoculation of sporozoites. Sporozoites actively invaded anterior end first, which resulted in extensive damage to the host cell. Such cells showed disrupted microvilli; protuberances of cytoplasm into the lumen, apparently the result of a disrupted plasma membrane; vacuolization of the cytoplasm; and damage to the mitochondria. These damaged cells were rapidly vacated as the sporozoite moved laterally into one or more adjacent intact host cells without entering the lumen. It is suggested that the host cell initially entered from the lumen becomes so severely traumatized that the parasite of necessity enters an adjacent cell as a prelude to further development. Various aspects of host-cell invasion by coccidia and malarial parasites are reviewed.

Animals↗

PubMed1992

Scanning and transmission electron microscopy of host cell pathology associated with penetration by Eimeria papillata sporozoites.

Scanning and electron microscopy was used to study the pathogenesis that occurred in mouse epithelial cells that had been penetrated by Eimeria papillata sporozoites. Optimal penetration of parasites injected into nonligated and ligated mouse intestine was found to occur at 4-15 min post-inoculation. During initial penetration, the parasite caused disruption of the microvilli of the intestinal cells, which led to detachment of the microvilli from the plasma membrane of the penetrated cell. Host cells penetrated by the parasite showed extensive destruction of the internal cellular organization together with blebbing of host-cell cytoplasm and release of internal organelles such as mitochondria. Ultimately, the penetrated cells completely broke down, leaving vacuolated areas next to ultrastructurally normal epithelial cells.

Animals↗

PubMed1992

Maternal immunization with gametocyte antigens as a means of providing protective immunity against Eimeria maxima in chickens.

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

Animals↗

PubMed1992

Effect of monensin on ultrastructure and cellular invasion by the turkey coccidia Eimeria adenoeides and Eimeria meleagrimitis.

Freshly excysted sporozoites (SZ) of the turkey coccidia Eimeria meleagrimitis and Eimeria adenoeides were incubated at 41 C in concentrations of monensin from .01 to 1.0 microgram/mL, washed free of the drug, and either processed for phase, fluorescence, and transmission electron microscopy or inoculated into cultures of turkey kidney cells. Phase microscopy indicated that after 1.5 h incubation in 1.0 micrograms/mL monensin, about 60% of the SZ of E. meleagrimitis had become notably rounded or displayed localized protrusions. These alterations were accompanied by ultrastructural abnormalities (in 90% of the SZ) including vacuoles in the cytoplasm, bulging and separation of plasma membrane layers, and dense bands in the refractile bodies that extended toward the periphery of the refractile body. Similar morphological and ultrastructural changes were observed in over half of the E. adenoeides SZ after 2 h incubation in 1.0 micrograms/mL monensin. Additionally, some specimens contained a pycnotic nucleus that was usually surrounded by a large vacuole. After 4 h incubation, almost all of the SZ displayed some degree of ultrastructural damage. Indirect fluorescent antibody labeling with parasite-specific monoclonal antibodies demonstrated clouds of antigen surrounding the monensin-treated but not the untreated SZ, suggesting an increase in permeability with incubation in monensin. With both E. meleagrimitis and E. adenoeides, the structural changes were reflected in a significant inhibition of cellular invasion. The inhibitory activity of monensin was concentration- and time-dependent in that the greatest inhibition of invasion was observed in SZ incubated for 4 h in 1.0 micrograms/mL of monensin; shorter incubation times or lower concentrations of monensin having less effect.

Animals↗

PubMed1992

Localization of a low molecular weight antigen of Eimeria tenella by use of hybridoma antibodies.

Three monoclonal antibodies (Mabs), found by western blot analysis to recognize 10-kDa bands of Eimeria tenella sporozoite preparations, were used with immunoelectron (IE) microscopy, immunogold-silver staining (IGSS), and indirect immunofluorescent antibody (IFA) light microscopy to determine the location and distribution of the antigens in or on extra- and intracellular parasites. All 3 of the Mabs (designated C3, E5, and 1231) were found by IE microscopy to label amylopectin granules of extracellular sporozoites. Additionally, these Mabs extensively gold-labeled the sporocyst wall. In cultured primary chicken kidney cells inoculated with sporozoites of E. tenella, IGSS showed surface labeling of the parasite and intense labeling of the infected host cells by 6 hr postinoculation (PI). At 24 hr PI, host cell vacuoles in infected and uninfected cells were labeled by the 3 Mabs by IFA. The E5 and C3 Mabs also were seen to label the host cell membrane of newly infected cells. The C3 and 1231 Mabs showed little label of the host cells by 48 hr PI, but the parasites still were labeled up to 96 hr PI. The E5 Mab had intense IFA labeling of infected host cells at 48 hr PI. The results of this study indicate that parasites apparently release antigenic material during the early stages of parasite development and that this material is found internally and/or on the surface of the infected host cells.

Animals↗

PubMed1991

Development of resistance to coccidiosis in the absence of merogonic development using X-irradiated Eimeria acervulina oocysts.

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

Animals↗

PubMed1991

Evolutionary relationships of avian Eimeria species among other Apicomplexan protozoa: monophyly of the apicomplexa is supported.

Direct, reverse transcriptase-mediated, partial sequencing of the small-subunit (16S-like) ribosomal RNA (srRNA) of Eimeria tenella and E. acervulina was performed. Sequences were aligned by eye with six previously published, partial or complete srRNA sequences of apicomplexan protists (Plasmodium berghei, Theileria annulata, Cryptosporidium sp., Toxoplasma gondii, Sarcocystis muris, and S. gigantea). Six eukaryotic protists (a slime mold, a yeast, two dinoflagellates, and two ciliates) acted as an outgroup for a parsimony-based phylogenetic analysis (PAUP Ver. 3.0). The 188 phylogenetically informative sites (i.e., those positions that neither were unvaried nor had only autapomorphic substitutions) supported a single tree topology 481 steps in length with a consistency index of 0.65 in which the monophyly of the Apicomplexa was supported. The two Eimeria species and S. muris, S. gigantea, and T. gondii formed a pair of monophyletic groups that were sister groups. The two Sarcocystis species were not hypothesized to be sister taxa. The genera Plasmodium and Cryptosporidium were hypothesized to form the sister group to these five coccidia and T. annulata. A priori data-editing techniques that deleted "variable" positions prior to analysis failed to recognize the monophyly of the Apicomplexa when the same parsimony-based tree-building algorithm was used. Inability of the outgroup taxa to root the well-supported ingroup tree (Apicomplexa) at a unique site when these taxa were used individually for this purpose reinforces the need for an appropriate, multiple-taxon outgroup in such analyses.

Animals↗

PubMed1991

X-irradiation of Eimeria tenella oocysts provides direct evidence that sporozoite invasion and early schizont development induce a protective immune response(s).

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

Animals↗

PubMed1991

Protective immunization against the intestinal parasite Eimeria acervulina with recombinant coccidial antigen.

The gene encoding an immunodominant Eimeria acervulina merozoite surface antigen (EAMZ250) was expressed in bacteria as a fusion peptide with the galactose-binding protein (GBP) of Escherichia coli. Recombinant and control antigens were administered to 1-wk-old chickens by peroral inoculation with live nonpathogenic bacteria that were expressing GBP-EAMZ250 or GBP protein. The immunization elicited antigen-specific humoral and cellular immune responses as measured by ELISA and T-cell blastogenesis assay. In addition, chickens immunized with recombinant GBP-EAMZ250 exhibited significant protection against weight loss and intestinal lesions after E. acervulina challenge. Bacterial transformants were recoverable from the upper and middle intestine of inoculated chickens for various times after immunization. These data indicate that oral administration of live E. coli expressing a recombinant E. acervulina antigen is an effective means of inducing resistance to coccidiosis.

Animals↗

PubMed1991

Characterization of a recombinant Eimeria acervulina antigen expressed in sporozoite and merozoite developmental stages.

A cDNA from Eimeria acervulina encoding an immunogenic region of antigens shared between sporozoites and merozoites was cloned and expressed in Escherichia coli. Immunofluorescence staining of sporozoites, merozoites, and coccidial-infected intestinal tissue with monoclonal antibody used to detect the recombinant clone indicated that the epitope was present on internal parasite proteins. Immunostaining of nitrocellulose paper containing protein from both asexual stages revealed numerous sporozoite antigens (18-120 kDa) and only 1 merozoite antigen (150 kDa). Northern blot hybridization assays using this cDNA clone for probing sporozoite and merozoite RNA showed that distinct transcripts were present in both asexual stages. Similar to the immunofluorescence studies, many homologous RNAs were observed in sporozoites (3.7-13 kb) and only 1 prominent hybridizing species was found in merozoites (3.3 kb). The recombinant coccidial antigen, designated MA16, is a 125-kDa beta-galactosidase fusion protein, representing about 10 kDa of parasite protein. In blastogenesis assays, purified recombinant MA16 antigen is capable of activating T lymphocytes obtained from E. acervulina-immune inbred chickens. DNA sequencing of MA16 clone and analysis of the predicted amino acid sequence indicated several putative T cell epitopes that may be responsible for the observed in vitro blastogenic response.

Amino Acid Sequence↗

PubMed1990

Eimeria acervulina: cloning of a cDNA encoding an immunogenic region of several related merozoite surface and rhoptry proteins.

A cDNA encoding a recombinant Eimeria acervulina antigen, designated EAMZp30-47, that contains an epitope shared among several surface and rhoptry proteins of merozoites was characterized. The respective parasite proteins are between 30 and 47 kDa as revealed by immunostaining of nitrocellulose membrane containing extracts of 125I-labeled merozoites. As indicated by immunofluorescence and immunoelectron microscopic staining, the reactive epitope was localized to both the surface membrane and the internal rhoptries of this asexual stage of the parasite. The recombinant beta-galactosidase fusion protein EAMZp30-47 is 130 kDa, thus representing 15 kDa or 30-50% of the respective parasite protein. Purified EAMZp30-47 stimulates T cells from E. acervulina-immune inbred chickens, but is not recognized by immune chicken serum, suggesting that T cell and not B cell epitopes recognized by the host immune system during a natural infection are present on the recombinant protein. Northern and Southern blot hybridization experiments indicated that expression of EAMZp30-47 is restricted to the merozoite stage of the parasite and the gene occurs as a single copy sequence within the genome.

Amino Acid Sequence↗

PubMed1989

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.

Animals↗

PubMed1989

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.

Amino Acid Sequence↗