Eimeria tenella: accumulation and retention of anticoccidial ionophores by extracellular sporozoites

Extracellular sporozoites of Eimeria tenella were incubated at either 25 or 40 C in the presence of ¹⁴C-lasalocid or ¹⁴C-narasin, both anticoccidial ionophores. Liquid scintillation analysis shows that both ionophores are accumulated by the sporozoites outside their host cell. The relative degree of retention was significantly different for the two incubation temperatures and the concentration of lasalocid retained was consistently greater than that of narasin.     

Eimeria tenella: Cecal contraction in infected chickens

The muscle of ceca from chickens infected with Eimeria tenella had an increased amplitude of contraction when compared to the cecal muscle of uninfected control chickens. The increased amplitude was significant (P ? 0.05) at 5 days postinoculation (PI) and became nonsignificant at 7 days PI. The amplitude also increased with the severity of the infection. The sensitivity of the muscle from infected ceca to acetylcholine (ACH) was greater than the control. Infection by different strains of E. tenella also increased the sensitivity of the cecal muscle to ACH when compared to the uninfected control. The rate of spontaneous contractions was not different in any group or treatment. The wet weight of infected ceca increased with days PI and severity of infection.     

Eimeria meleagrimitis, E. adenoeides, and E. dispersa: Severity of infection and changes in the intestinal mucosa of the Turkey

Glucose and methionine were malabsorbed in some intestinal regions of turkeys infected with Eimeria meleagrimitis, E. adenoeides, or E. dispersa. The decrease in absorption was not always related to the numbers of parasites in the cells or the extent of damage to the mucosa. With E. adenoeides, malabsorption was found in the jejunum even though parasites were not present. Conversely, with E. dispersa, no malabsorption was observed in the duodenum even though light microscopy showed numerous parasites. In many intestinal regions, damage to the mucosal surface visible with scanning electron microscopy (SEM) was slight or absent, although malabsorption was marked. No changes were noted with SEM in the structure and orientation of the brush border in these regions. Villar height was significantly reduced in the regions of heaviest infection when intestinal damage was visible. Conversely, the crypts of Lieberkühn were often two or three times as deep in infected poults as in uninfected poults. In general, no differences were found in the thickness of the circular and longitudinal muscle layers between the infected and uninfected poults. The dry weight of the intestinal tissue was less from infected poults than from uninoculated controls and was related to both region of the intestine and severity of the infection.     

Eimeria nieschulzi and Trichinella spiralis: Analysis of concurrent infection in the rat

The effects of concurrent primary infection of the rat with Eimeria nieschulzi and Trichinella spiralis on the number of oocysts of E. nieschulzi shed by the host and on the number, distribution, and fecundity of adult T. spiralis were analyzed. When rats were initially infected with E. nieschulzi followed 9 days later by infection with T. spiralis there occurred a significant decrease in the total numbers of adult worms in the small intestine, a significant shift in the position of these worms along the length of the small gut, a decrease in the fecundity of adult female worms, and a decrease in muscle parasitism when compared with rats infected with T. spiralis alone. When rats were initially infected with T. spiralis, followed 9 days later by infection with E. nieschulzi, there occurred a significant decrease in the numbers of oocysts shed over 24 hr on Days 7, 9, and 11 postinfection below that seen with rats infected only with Eimeria. These changes are discussed in terms of the enteropathophysiologic lesions and enteric inflammation known to occur during infections with these two parasites.     

Eimeria necatrix: Induced proteolytic sensitivity of infected chick crypt cells

While devising a protocol for the isolation of chick crypt cells infected with Eimeria necatrix, it was observed that infected cells were readily lysed by 0.25% trypsin. Time-course studies at 17 C with 5.5 × 10⁵ cells at 96 hr postinfection revealed that 0.001% trypsin effectively lysed >90% of infected cells within 10 min. Uninfected crypt cells were not lysed under these conditions. To determine the site of action of trypsin, the plasma membrane proteins from trypsin-treated and untreated infected cells were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. While the major proteins were unaffected by the trypsin treatment, some minor changes were noted: (1) three components (～-60, ～-52, and ～-20 KDa) were trypsin sensitive and (2) a new band (～-42 KDa) appeared in the membrane of trypsin-treated infected cells. Previously, it was found that the plasma membrane of infected cells, in contrast to uninfected cells, accumulated gel-phase lipid (J. E. Thompson, M. A. Fernando, and J. Pasternak, Biochimica et Biophysica Acta555, 472–487, 1979). Here, it was examined whether trypsin would perturb the physical state of the plasma membrane of infected cells. Both X-ray diffraction patterns and transition temperature studies revealed no difference between membranes from untreated and trypsin-treated infected cells. Thus, “trypsin sensitivity” may be a secondary phenomenon that is due primarily to the cellular leakiness that accompanies the accumulation of gel-phase lipid in the plasma membrane of infected cells. The uptake of trypsin may stimulate the release of catabolic enzymes that, consequently, lyse an infected cell.     

Eimeria tenella,E. necatrix,E. acervulina,and E. maxima: Anticoccidial activity of 1,6-dihydro-6-oxo-2-pyrazinecarboxylic acid 4-oxide

The anticoccidial activity of an orotic acid analog, 1,6-dihydro-6-oxo-2-pyrazinecarboxylic acid 4-oxide (carboxyemimycin), was tested in battery experiments, utilizing 9-day-old Single-Comb White Leghorn cockerels. Carboxyemimycin, at 125 ppm and more in feed, exhibited marked anticoccidial activities against Eimeria tenella, E. necatrix, E. acervulina, and E. maxima. High doses of carboxyemimycin—up to 1000 ppm—did not cause any reduction in weight gains. The battery and in vitro studies with delayed and restricted medications revealed that carboxyemimycin affected the development of E. tenella in first and second generation schizogony and in gametogony.     

Plasmodium berghei and Plasmodium knowlesi: Serum binding to sporozoites

Plasmodium berghei salivary gland and oocyst sporozoites were examined with fluorescein isothiocyanate (FITC)-lectins to determine if sporozoites had carbohydrate-containing molecules on their surfaces. None of the eight fluorescein isothiocyanate-lectins bound to the sporozoites. However, incubation of sporozoites in mouse serum permitted subsequent binding of concanavalin A and Ricinus communis agglutinin I. In general, serum binding occurred when sporozoites were incubated in serum from hosts susceptible to sporozoite infection. Sporozoites of the rodent parasite, P. berghei, tended to bind rodent but not primate serum, while sporozoites of the monkey parasite, Plasmodium knowlesi, tended to bind primate but not rodent serum. The serum component(s) that bound to sporozoites were concentrated considerably by ammonium sulfate precipitation followed by concanavalin A—Sepharose affinity chromatography.     

The influence of the fatty acid composition of Acholeplasma laidlawii membranes on the growth inhibitory activity of narasin,a polyether ionophorous antibiotic

Narasin, a polyether ionophorous antibiotic capable of acting as a transmembrane carrier of cations, has a growth inhibitory effect on Acholeplasma laidlawii, permitting only 20% survival when present at 0.1 μg/ml in an undefined growth nutrient or fatty acid-deficient nutrient supplemented only with palmitic acid. When A. laidlawii is propagated in fatty acid-deficient nutrient supplemented with linoleic acid, however, the organisms become 40 times more sensitive to the growth inhibitory effect of this antibiotic. The actual fatty acid compositions of the membranes would indicate that a higher degree of unsaturation enhances ionophore activity.     

Eimeria Spp.: Influence of coccidia on digestion (amylolytic activity) in broiler chickens

Loss in pancreatic weight and an overall decrease in amylolytic activity in the pancreas were seen in broiler chickens infected with Eimeria acervulina, E. maxima, and E. necatrix. Only E. acervulina and E. maxima reduced amylolytic activity in the surface mucosa of the regions they infected. Surface-bound amylolytic activity decreased as the severity of infection (as measured by lesion score) with E. acervulina increased. This decrease in activity was not related to the decreased feed consumption in infected birds. Little decrease in amylase activity was found in the lumenal contents with the exception of E. acervulina in the jejunum. When the effect of pH on enzyme activity was studied in vitro, a marked reduction in amylolytic activity was observed as the pH went below 5.0.     

Plasmodium lophurae: Antibody-induced movement and capping of surface membranes of erythrocyte-free malarial parasites

Surface antigens of the avian malarial parasite, Plasmodium lophurae, and its host cell, the duckling erythrocyte, were visualized at the ultrastructural level using rabbit antisera and ferritin-labeled goat anti-rabbit IgG. Rabbit antisera to P. lophurae caused an aggregation of parasite and parasitophorous vacuole surface membrane antigens, a phenomenon known as capping. Capping required living plasmodia and did not occur if parasites had been fixed with glutaraldehyde prior to exposure to antisera. Antisera against duckling erythrocytes did not cross-react with erythrocyte-free malarial parasites, and did not form caps on the surface of the red blood cell. Antiplasmodial sera did not react with normal or malaria-infected red cells. These results suggest that surface membrane proteins of the intracellular plasmodium are capable of lateral movement.     

Toxoplasma gondii: Calcium lonophore A23187-mediated exit of trophozoites from infected murine macrophages

The Ca²⁺ ionophore A23187 consistently induced the exit of Toxoplasma gondii trophozoites from cultured macrophages which they had recently infected. Following exit of toxoplasmas, the host macrophages underwent degeneration. A23187 was active at concentrations higher than 0.25 μM and the activity reached a plateau at the concentration of 1.0 μM. Noninfected macrophages or those engulfing heat-killed toxoplasmas, or some other particles, were not affected by treatment with A23187. The toxoplasmas exiting host cells were capable of infecting and proliferating in normal macrophages. The A23187-mediated exit of toxoplasmas proceeded despite external Ca²⁺ and was enhanced by the addition of ethylene glycol bis(β-aminoethyl ether) N,N,N′,N′-tetraacetic acid (EGTA) in the reaction mixture. On the other hand, the A23187-mediated exit of toxoplasmas was inhibited significantly by exogenous Mg²⁺.     

Trypanosoma cruzi: Isolation and characterization of a proteinase

Lysates of Trypanosoma cruzi epimastigotes were able to hydrolyze casein (K_m = 2.5 mg/ml) as well as bovine and human hemoglobins (K_m = 12.2 mg/ml); there was optimum activity was around pH 7.0. The proteinase activity detected with these substrates was enhanced by sodium diaminotetraacetate (EDTA) and reducing agents (SO^2?₃, mercaptoethanol, cysteine) and was inhibited by sulfhydryl reagents, thus suggesting an SH-dependent enzyme. Purification (60×) of the proteinase was carried out as follows: (1) precipitation at ?20 C, pH 4.5, with 80% acetone, (2) gel filtration on Sephadex G-200, (3) affinity chromatography on Sepharose 4B covalently linked to p-aminophenyl mercuric acetate. Only a single component (with an estimated molecular weight of 60,000) was detected in purified preparations by polyacrylamide gel electrophoresis. However, in addition to the major component identified as a proteinase, crossed immunoelectrophoresis experiments indicated the presence of at least three other antigens that apparently were devoid of proteinase activity. Optimum pH activity of the purified preparations was around pH 6.0 for casein and pH 3.0 for hemoglobins, but these activities probably are due to the one enzyme since they were altered identically by the same agents.     

Leishmania tropica: Surface antigens of intracellular and flagellate forms

Promastigotes and amastigotes of Leishmania tropica were surface-radioiodinated using the lactoperoxidase technique. Detergent lysates of the labeled organisms were analyzed by two-dimensional gel electrophoresis. Analysis of radioiodinated promastigote membrane proteins revealed six major and some minor acidic polypeptides. Analysis of the amastigote membrane proteins revealed six major proteins, mostly acidic, and some poorly resolved basic proteins. Four of the major membrane proteins appeared to be common to the two parasitic forms (M_r 67,000, M_r 50,000, M_r 68,000, and M_r 80,000). These polypeptides were recognized by antipromastigote antibodies as well as antibodies from CBA/H mice that had recovered from infection. Peptide mapping confirmed their homology in the two parasite forms. One polypeptide appeared to be specific for the promastigote (M_r 50,000) and two polypeptides appeared to be specific for the amastigote form of the parasite (M_r 94,000 and M_r 43,000).     

Tritrichomonas foetus: Localization of filipin-sterol complexes in cell membranes

The polyene antibiotic, filipin, was used as a probe for the detection of sterols in the freeze-fractured plasma membrane and the flagellar membranes of the pathogenic protozoa, Tritrichomonas foetus. A homogeneous distribution of filipin-sterol complexes was seen throughout the plasma membrane, and the membrane of the three anterior and the one recurrent flagella. No or very few filipin-sterol complexes were observed in some specialized regions such as the base of the flagella (necklace), the portion of the recurrent flagellum, and that part of the cell body to which the flagellum was attached. The density of filipin-sterol complexes varied from one cell to the other. In some cells, about 205 complexes/μm² were seen. A larger number of filipin-sterol complexes were observed on both faces of the membrane of cytoplasmic structures, probably corresponding to vacuoles. No complexes were seen in the nuclear membrane and in the membrane of the endoplasmic reticulum. Very few or no complexes were observed in the membrane of the hydrogenosomes. Treatment of living cells with filipin induced aggregation of filipin-sterol complexes at some points of the plasma membrane.     

Toxoplasma gondii: Microassay to differentiate toxoplasma inhibiting factor and interleukin 2

Using a sensitive, economical, and reproducible microassay, the relationship of toxoplasma inhibiting factor to interleukin 2 has been examined. The assay developed took advantage of the observation that (1) Toxoplasma gondii tachyzoites replicated efficiently in the murine monocytic cell line, RAW 264; (2) treatment of RAW 264 cells with toxoplasma inhibiting factor prevented intracellular replication of the parasite to an extent similar to that observed with identical treatment of freshly isolated murine peritoneal exudate cells; and (3) [³H]uracil incorporation was an efficacious means to quantify replication (or inhibition of replication) of tachyzoites within the cell line. Although toxoplasma inhibiting factor and interleukin 2 were both present in the same lectin -and antigen-stimulated splenocyte supernatant fluids, results from microassays strongly suggested that the molecules were two distinct entities.     

Eimeria tenella,Eimeria necatrix, and Eimeria adenoeides: Peripheral blood leukocyte response of chickens and turkeys to strains adapted to the turkey embryo

The peripheral blood leukocyte responses of chickens and turkeys inoculated with one of three strains of a chicken Eimeria species adapted to the turkey embryo with their respective parent lines, or with E. adenoeides of the turkey were studied. The adapted lines tended to cause hematological changes in chickens and turkeys similar to those caused by E. adenoeides. These parasites caused the most significant increases in large mononuclear white blood cells = (monocytes) in both chickens and turkeys. These results provide further evidence for a monocyte/macrophage effector mechanism in the rejection of heterologous species of Eimeria from a nonspecific host. The results also agree with previous studies that show that increases in mononuclear white blood cells during parent E. tenella and E. necatrix infections in chickens occur during the periods of greatest tissue damage (3–4 days after inoculation). The generally unaffected lymphocyte numbers and increases in mononuclear white blood cells during infections with the adapted lines probably explain the reduced pathogenicity and the lack of immunogenicity seen previously in chickens inoculated with these three lines. Possibly, monocytes/macrophages play a role in the host specificity of the parasites.     

Schistosoma haematobium: Amoscanate and adult worm ultrastructure

Amoscanate, when administered orally as an aqueous or “formulated” preparation, induced pronounced ultrastructural abnormalities in male and female Schistosoma haematobium. Higher dose levels of the aqueous suspension (300 mg/kg body wt) had to be administered to achieve the full range of effects induced by formulated doses of 2.5–8 mg/kg body wt. Worms were recovered from hamsters between 1 and 120 hr after treatment. Although the amount of amoscanate-induced damage varied considerably between worms, an overall pattern of damage emerged. Initially, 1 hr after treatment, amoscanate caused tegumental vacuolation and oedema. As the drug treatment period was extended to 24 hr, blebbing, exudation, collapse of sensory organelle bases, and abnormal mitochondria became increasingly evident. With exposure to higher drug doses (50–300 mg/kg body wt), the tegument became further distorted with the appearance of necrotic structures and myelin whorls, which appeared to represent various stages in lysosomal formation and digestion. Eventually, erosion of surface layers resulted in the breakdown of tegumental integrity. The caeca and vitellaria were also adversely affected by drug treatment. Basal vacuolation and the formation of myelin whorls occurred in the gastrodermis. In the mature S4 vitelline cells, coalesced vitelline droplets and myelin whorls were evident.     

Trypanosoma cruzi: Effect on B-cell-responsive and -responding clones

During the course of Trypanosoma cruzi infection in C57BL/6 mice, which are relatively resistant to the parasite, the hosts developed antibody activity against previously unencountered antigens. The anti-sheep erythrocyte and antitrinitrophenyl antibody levels increased rapidly from Day 7 of infection, reached a peak by the 21st day, and were maintained at this level through 120 days postinfection in these mice. In contrast, highly susceptible C3H(He) mice did not have demonstrable antibody responses to SRBC or TNP during the 24-day infection period. Autoantibody activity against the selfantigens presented on isologous erythrocytes or thymocytes, however, were reduced in infected C57BL/6 mice. No significant reduction in autoreactivity to the self-antigens on erythrocytes or thymocytes was observed in C3H(He) mice infected with T. cruzi although a trend of reduced autoresponsiveness toward erythrocytes appeared to be developing by the time of death. C57BL/6 mice immunized with sheep erythrocytes as neonates and infected with T. cruzi as adults, or adult mice primed with low doses of sheep erythrocytes prior to infection, had elevated antibody responses to sheep erythrocytes unless the mice were immunized with sheep erythrocytes during the course of infection, in which case suppression of the response against sheep erythrocytes resulted. The nonspecific synthesis of immunoglobulins in infected C57BL/6 mice was, in part, a result of the lymphocyteactivating properties of T. cruzi-associated antigens. The T. cruzi-associated antigens induced proliferative and differentiative responses in spleen cells in vitro. It is proposed that the T. cruzi-associated antigens differentially affect lymphocytes capable of responding to antigen and those lymphocytes previously stimulated by antigen.     

Plasmodium berghei: Electron spin resonance and lipid analysis of infected mouse erythrocyte membranes

Red blood cells from mice infected with Plasmodium berghei and from uninfected mice were labeled with stable, free radical derivatives of stearic acid. Electron spin resonance spectra of these samples showed that the degree of molecular order in these membranes decreased, and the rate of motion of the probe increased, with increasing levels of parasitemia. Parasitemia increased the ratio of unsaturated to saturated 18-carbon fatty acids, and decreased the percentage of arachidonic acid and of cholesterol. The effects of parasitemia on the membrane properties correlated with decreases in cholesterol/fatty acid ratios.     

Trypanosoma equiperdum: Movement from the dermis

Twelve dogs were injected intradermally with 352,770 to 14,391,660 Trypanosoma equiperdum and afferent and efferent lymph, lymph nodes, and blood examined by mouse inoculation at minute, hourly, and daily intervals following inoculation. The log dosage of trypanosomes given each dog was closely related to their body weight (P < 0.01). Afferent lymph contained trypanosomes as soon as 5 and 27 min after inoculation. Lymph nodes on the side of injection became positive within 5 min of injection, while those on the contralateral side remained negative for at least 120 min after injection. Blood contained trypanosomes as soon as 5 min after injection, although the average time for all dogs, before trypanosomes were demonstrated in the blood, was 40 min postinjection. Efferent lymph did not contain organisms until 25–76 hr after inoculation. We consider this sequence to indicate that T. equiperdum can leave the dermis in afferent lymphatics, reach the local lymph node, invade the blood stream from this site, and only after a day or longer do they leave the node via the efferent lymphatics.