Biomedical subjects

H D Danforth

Publications and source records attributed to H D Danforth.

At least 55 records · Page 3Linked to original sources

PubMed1989

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.

Animals↗

PubMed1989

Major histocompatibility complex control of immunity elicited by genetically engineered Eimeria tenella (Apicomplexa) antigen in chickens.

The immunogenicity of a recombinant Eimeria tenella coccidial antigen was studied in 6(1).B congenic chickens derived from B2B2 and B5B5 parents segregating for haplotypes B2 and B5. Five-week-old chickens were immunized with 2.4 micrograms of recombinant protein (designated 5401) in Freund complete adjuvant and challenged with 75,000 oocysts at 28 days postimmunization (DPI) to determine the degree of elicited protective immunity. Serum samples were collected weekly for 5 weeks postimmunization for analysis by enzyme-linked immunosorbent assay, immunofluorescence assay, and Western blotting. Lesion scores following oocyst challenge were significantly reduced in B5B5 chickens compared with those in B2B2 chickens. Immunization induced a sporozoite-specific immunoglobulin G (IgG) titer in serum detected by the enzyme-linked immunosorbent assay that peaked at 28 DPI, the day of challenge, in B5B5 chickens and at 42 DPI in B2B2 chickens. After challenge, this titer declined for each genotype. Anti-sporozoite IgG detected by the immunofluorescence assay attained a peak titer at 21 DPI in B2B2 chickens and 28 DPI in B5B5 chickens. Serum from immunized B5B5 chickens reacted strongly in Western blots with several high-molecular-weight (greater than 100,000), soluble proteins prepared from sporozoites. Serum from B2B2 chickens reacted with similar proteins as well as with a 51- to 53-kilodalton protein that was not labeled by serum from B5B5 chickens. These results demonstrate further the role of host genetics on anticoccidial immunity and suggest that a peak anti-sporozoite IgG titer in B5B5 chickens on the day of challenge may signal a state of immunocompetence to that challenge.

Animals↗

PubMed1989

Genetically engineered antigen confers partial protection against avian coccidial parasites.

A fusion protein of beta-galactosidase and Eimeria tenella produced in a recombinant Escherichia coli strain was injected into chickens and elicited partial protection against an oral challenge with Eim. tenella parasites. The fusion protein contained a 31 kilodalton (kD) coccidial antigen designated as 5401. The DNA sequencing of the 5401 antigen-coding sequence revealed that this protein segment was highly negatively charged and strongly hydrophilic, and contained an amino-acid sequence repeated five times. A dose-titration study showed that immunizing chickens with a single subcutaneous injection of the 5401 antigen at 1,200 to 4,800 nanograms (ng)/bird in Freund's complete adjuvant decreased lesion scores, mortality, and feed conversions compared to unimmunized, challenged controls. Using the 1,200 and 2,400 ng/bird of the 5401 antigen, group weight gains were higher than for the unimmunized, challenged birds. In three other trials using the 5401 antigen at 2,400 ng/bird with light, medium, and heavy coccidial infections, significant protection was evidenced by reduced lesion scores, increased individual weight gains, or both. In addition, feed conversions were reduced when compared with unimmunized controls or birds immunized with a noncoccidial protein E. coli extract. Western blot analysis of sporozoite preparations with serum from 5401-immunized birds labeled two antigenic bands of 66 and less than 200 kD. These results indicate that the coccidial proteins produced in E. coli are potentially effective immunogens for protecting chickens against avian coccidiosis.

Amino Acid Sequence↗

PubMed1988

Monoclonal antibodies reveal antigenic differences in refractile bodies of avian Eimeria sporozoites.

Monoclonal antibodies were developed against refractile body antigens of 4 species of avian Eimeria, E. meleagrimitis, E. adenoeides, E. acervulina, and E. tenella. Although antibodies from 8 different cell lines were used in this study, all produced similar fluorescent and gold-labeling patterns. By immunofluorescent antibody techniques, 5 of the 8 antibodies cross-reacted with all 4 of the Eimeria species that were examined; the other 3 antibodies reacted only with the species against which they were produced or with a limited number of species. In Western blot analyses using SDS-solubilized sporozoites as antigen, 4 of the cross-reactive antibodies recognized multiple bands; the predominant bands had molecular weights of approximately 23, 45, and 90 kilodaltons (kDa). Two of the antibodies with more limited reactivity recognized either a single band at 23 kDa (91C7), or bands at 23 and 45 kDa (4115); another reacted only with several bands greater than 100 kDa (4D10). The molecular weights of the antigens did not decrease markedly after digestion with N-glycanase F, indicating that if the refractile body antigens contained significant amounts of N-linked carbohydrate it was refractory to the enzyme. Collectively, the data indicate that antigens of the sporozoite refractile bodies differ among the Eimeria species. Some antigens are conserved, whereas others differ in distribution or frequency among the individual species.

Animals↗

PubMed1987

Effect of ionophorous anticoccidials on invasion and development of Eimeria: comparison of sensitive and resistant isolates and correlation with drug uptake.

Prophylactic levels of three ionophorous antibiotics, monensin, salinomycin, and lasalocid, were administered to groups of chickens and turkeys. All three ionophores markedly inhibited invasion of cecal tissues by sporozoites of ionophore-sensitive (IS) Eimeria tenella. Monensin and salinomycin also reduced invasion in turkeys by sporozoites of E. adenoeides, but lasalocid only minimally inhibited invasion. Invasion of ceca of monensin-medicated chickens was significantly greater by sporozoites of ionophore-resistant (IR) E. tenella than of the IS isolate. Concomitant experiments showed significant differences in [14C]monensin accumulation among IS and IR isolates of E. tenella. The decreased uptake of monensin by the IR isolates appeared to be accompanied by a decrease in responsiveness to the activity of monensin as well as to two other ionophores, salinomycin and narasin in cell culture. The amount of monensin, salinomycin or narasin required to inhibit development of E. tenella by 50% was 20 to 40 times higher for the IR isolates than for the IS ones. Collectively, the data suggest that differences in ionophore accumulation by IS and IR isolates of E. tenella might reflect differences in membrane chemistry and that these differences are responsible for the expressions of resistance that were observed in these studies. This expression of resistance appears to be common to all ionophores tested.

Animals↗

PubMed1987

Hybridoma antibody characterization of stage-specific and stage-cross-reactive antigens of Eimeria tenella.

Hybridoma antibodies (HAb) have been raised against the sporozoite stage of 3 species of avian coccidia. These HAb were utilized in Western blot analysis, resulting in the immunoenzymatic detection of sporozoite and merozoite antigens of 1 species, Eimeria tenella. The 5 HAb specific for the sporozoite stage showed either single bands at 22 and 28 kDa or a large diffuse band in the 7-10-kDa range. The 4 HAb that cross-reacted with both asexual stages recognized either a single sporozoite or merozoite antigen of 90 kDa, or multiple antigens (47-69 kDa) for both stages. The 9 HAb demonstrated 5 different immunofluorescent antibody (IFA) patterns, and the 4 cross-reactive HAb showed similar IFA patterns with both asexual stages of E. tenella. The sporozoite-specific HAb which identified the 22, 7-10, and 7-8 kDa antigens showed surface, surface-internal, or internal IFA patterns. The other sporozoite-specific HAb, which labeled the 28-kDa antigen, stained the refractile body. The IFA of the 4 stage-cross-reactive HAb, which recognized the 45-60-kDa and the 90- or 47-69-kDa antigens, localized these antigens to the surface and tip, respectively. Rabbit anti-sporozoite serum appeared to recognize all of the sporozoite and merozoite antigens identified by the HAb as well as a variety of additional stage-cross-reactive antigens.

Animals↗

PubMed1984

Cellular pathology in mouse embryonic brain cells following in vitro penetration by sporozoites of Eimeria papillata.

The pathology that occurs in mouse embryonic brain ( MEB ) cells that have been penetrated by sporozoites of Eimeria papillata was studied by light and electron microscopy. At the light microscopy level the greatest number of intracellular parasites was seen at 15 and 45 min postinoculation (PI). The monolayer of MEB cells had begun to round up by 45 min PI, and by 60 min PI most of the cells were stripped from the coverslip. Little ultrastructural damage was seen in MEB cells just penetrated by the parasites at 15 min PI, and no host cell membrane was seen around the sporozoites that had just entered the cells. Flexing and bending of the sporozoites within the MEB cell caused vacuolization of cell cytoplasm and in some cases rupture of host cell membrane. Sporozoites leaving the host cells at 15 min PI caused a rupture of the host cell membrane at the apical end of the parasite, and both host cell membrane and cytoplasm were attached to the surface of the parasite. MEB cells still attached to coverslips at 45 min PI demonstrated complete degeneration.

Animals↗

PubMed1984

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.

Antibodies, Monoclonal↗

PubMed1984

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.

Animals↗

PubMed1984

Effects of cationized ferritin and neuraminidase on invasion of cultured cells by Eimeria meleagrimitis sporozoites.

Primary turkey kidney cells and Eimeria meleagrimitis sporozoites were treated with cationized ferritin (CF) or neuraminidase ( NANase ), and the effects on the invasion of the cells by the sporozoites were measured. Cultures of host cells pretreated with either compound contained significantly fewer intracellular sporozoites than did control cultures. There was little additive effect if cultures were first treated with NANase and then with CF. In contrast, pretreatment of sporozoites with CF or low concentrations of NANase had no effect on invasion. The inhibition of invasion was apparently due to an interaction between treatment substances and host cell surface rather than to direct effect on the sporozoites. The CF bound to the randomly distributed anionic sites on the surfaces of both host cells and sporozoites and then rapidly aggregated. Sporozoites, probably in the process of invading cells, were invariably found with the conoid in close association with aggregates of CF on the host cell membrane. The CF on the sporozoites was apparently shed before or during invasion because all intracellular sporozoites were completely devoid of the label.

Animals↗

PubMed1983

Specificity and crossreactivity of immune serum and hybridoma antibodies to various species of avian coccidia.

The species-specificity and crossreactivity of serum antibodies (Ab) from birds immunized specifically with six different species of coccidia and of 24 hybridoma antibodies (Ab) developed against four species of chicken and two species of turkey coccidia were determined by use of the indirect immunofluorescent antibody (IFA) test on air-dried sporozoites. With few exceptions, the immune chicken sera were found to crossreact with all species of coccidia tested. Seven of the hybridoma Ab were species-specific, while the other 17 Ab demonstrated varying degrees of crossreactivity. Similar types of IFA patterns were seen with both the species-specific and crossreactive hybridoma Ab. Some of the crossreactive hybridoma Ab produced one type of IFA pattern with the sporozoites against which they were originally raised and different patterns with other species of sporozoites. The development of the hybridoma Ab has made it possible to identify the species of coccidia found in mixed infections and check the purity of laboratory strains.

Animals↗

PubMed1983

Free-flow electrophoretic separation of Plasmodium berghei sporozoites.

Sporozoites of the rodent malaria, Plasmodium berghei, were obtained from infected Anopheles stephensi by grinding mosquitoes, prepurifying the material in a discontinuous Hypaque gradient and further purifying by means of continuous free-flow electrophoresis. Bacteria, debris, mitochondria, mitoplasts, and other contaminants were removed in the electric field. The isolated sporozoites were morphologically intact and were positive in indirect immunofluorescence assay. They were infective to mice prior to and following free-flow electrophoretic separation. The surface of the sporozoites exhibited a polysaccharide-rich layer. The predominant surface protein labelled after surface iodination had a molecular weight between 42,000 and 46,000 daltons.

Animals↗

PubMed1983

Use of monoclonal antibodies directed against Eimeria tenella sporozoites to determine stage specificity and in vitro effect on parasite penetration and development.

Three different hybridoma-produced monoclonal antibodies (Ab) were used to study their reactivities with in vitro developmental stages of Eimeria tenella and their effects on sporozoite penetration and intracellular development. One Ab (designated B10) was stage-specific, whereas the other 2 Ab (designated C3 and E5) reacted with various intracellular developmental stages of the coccidia. The E5 Ab interacted with the cytoplasm of cultured cells that were infected with sporozoites at 24 hours after inoculation. All 3 Ab inhibited penetration to various degrees--the one designated B10 having the greatest inhibitory effect. These 3 Ab also inhibited development of the parasite in cell culture, provided that Ab was continuously present in the cell culture medium. Removal of Ab from the medium allowed coccidial development to continue at about the same rate as in controls. A longer pretreatment time of the sporozoites with the Ab before cell inoculation increased the inhibitory effect with respect to both the penetration and the development of the parasite.

Age Factors↗

PubMed1982

Surface changes induced by immune serum on Eimeria tenella sporozoites and merozoites.

The surface of merozoites and sporozoites of Eimeria tenella was affected by incubation with E. tenella-immune chicken serum (ICS). Normal chicken serum (NCS) and heat-inactivated ICS had no effect on the pellicular surface of either developmental stage. Sporozoites formed surface bulges or swellings after 10 min of incubation with ICS, and by 15 min postincubation, the morphology of the sporozoites was distorted by a surface coating of fibrinous material. Merozoites exposed to ICS were similarly coated, but surface swelling was not as severe. The coating formed rapidly and was seen as early as 5 min postincubation. Sporozoites incubated with heat-inactivated ICS supplemented with normal chicken serum were coated with a fibrinous material and in some cases lysed. These data indicated that complement must be present for the surface interaction to occur.

Animals↗

PubMed1982

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.

Animals↗

PubMed1982

Production of monoclonal antibodies by hybridomas sensitized to sporozoites of Plasmodium berghei.

Hybridoma cell lines, which secreted antibodies directed against either the surface of Plasmodium berghei sporozoites or mosquito debris, were produced by fusion of spleen cells of P. berghei sporozoite-immunized mice with P3-X63-Ag8 myeloma cells. Four cloned antibody-secreting cell lines were successfully established. Two of these clones (F9 and G10) were obtained from the fusion of spleen cells from mice that had undergone two immunizations. These clones produced an IgM antibody that did not produce a CSP response on the sporozoite and did not neutralize the infectivity of the sporozoite in mice. One clone (B6), produced by the fusion of spleen cells from mice that had been given three immunizations, secreted an IgG antibody that did produce a CSP response on the sporozoite and neutralized the infectivity of the parasite. A fourth clone (D5) directed against mosquito debris was also found to produce an IgG antibody, but did not show a CSP reaction or neutralization of the sporozoite infectivity.

Animals↗

PubMed1982

Development of hybridoma-produced antibodies directed against Eimeria tenella and E. mitis.

Hybridoma cell lines, which secreted antibodies directed against two different strains of Eimeria tenella and one strain of E. mitis, were produced by fusion of spleen cells from sporozoite-immunized Balb/cByJ mice with P3-X63-Ag8 myeloma cells. The antibodies demonstrated at least eight different binding patterns on or in air-dried sporozoites as determined by the indirect immunofluorescent antibody (IFA) test. These patterns varied from a general internal fluorescence similar to that seen with sporozoites exposed to hyperimmune chicken serum, to fluorescence observed on the tip, pellicle, and refractile body of the parasite. Five cloned, antibody-secreting cell lines were successfully established. Four of these clones produced antibody that reacted only with various strains of E. tenella and cross-reacted with no other species of coccidia. The fifth clone produced antibody directed against only E. mitis and did not react with any other coccidial species.

Animals↗

PubMed1980

Sporozoites of mammalian malaria: attachment to, interiorization and fate within macrophages.

Sporozoites of Plasmodium berghei and Plasmodium knowlesi, incubated in normal serum readily interact with peritoneal macrophages of mice or rhesus monkeys, respectively. Interiorization of the sporozoite requires that both serum and macrophages be obtained from an animal susceptible to infection by the malaria parasite. Serum requirements for sporozoite attachment to the macrophage are less specific. Phagocytosis is not essential for the parasites to become intracellular. Our findings indicate that active penetration of the sporozites into the macrohages does occur. Antibodies present in the serum of sporozoite-immunized mice are important in determining the fate of both the intracellular sporozoites and the macrophages containing the parasite. Sporozoites coated with antibodies degenerate within vacuoles of the macrophages, which have no morphologic alteration. Sporozoites incubated in normal serum do not degenerate within macrophages, but the parasitized macrophages become morphologically altered and are destroyed. Preliminary experiments indicate that sporozoites appear to interact with rat Kupffer cells in the same way as with the peritoneal mouse macrophages. It is postulated that Kupffer cells play a dual role in sporozoite-host cell interaction. In normal animals these cells might serve to localize the sporozoites in the immediate vicinity of the hepatocytes. In the immunized animals, macrophages would remove and destroy the antibody-coated parasites, thus contributing to sporozoite-induced resistance.

Animals↗