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 tenella: Effect of narasin,a polyether antibiotic on the ultrastructure of intracellular sporozoites

Eimeria tenella sporozoites were inoculated into cultures of chick kidney cells in the presence of 0.01 or 0.1 μg/ml of narasin and incubated at either 40 or 30 C for 24 hr. Electron microscopic examination revealed that either concentration of this polyether ionophore caused extensive ultrastructural damage to the intracellular sporozoite at 40 but not at 30 C, indicating that the severity of the coccidiocidal effect is influenced by temperature. The effect of 0.01 μg/ml monensin on the intracellular parasite was similar to that of narasin, suggesting a common destructive mechanism. The host cells were unaffected by 0.01 μg/ml of narasin at either temperature and by 0.1 μg/ml at 30 C, indicating that the polyether ionophores can be selectively lethal for the parasite. However, when the host cells were treated with 0.1 μg/ml narasin and incubated at 40 C, ultrastructural abnormalities were evident. The results suggest that the coccidiocidal effect of the polyether ionophorous antibiotics may be a general osmotic phenomenon.     

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.     

Giardia muris: Correlation between oral dosage,course of infection,and trophozoite distribution in the mouse small intestine

Oral inoculations of Giardia muris cysts to CD-1 Swiss mice resulted in a reproducible pattern of infection measured by cyst excretion and the number of trophozoites in the small intestine. Housing of animals together or individually did not alter the pattern of cyst release for the first 30 days of infection. Administration of 10 to 10⁵ cysts/mouse resulted in a similar rate of cyst excretion from Day 10 to the end of the infection period. There was a direct correlation between the size of inoculum and the length of the latent period. Mice which received larger doses (10³, 10⁴, 10⁵) had significantly higher numbers of cysts in feces during the first week of infection, compared to those which received lower doses (10 or 100). The extent of trophozoite colonization and their distribution in the small intestine was not related to the size of inoculum. The location of trophozoites in the small intestine varied during the infection. In the first 3 to 8 days of infection the trophozoites were situated in the upper 25% of the small intestine, but moved posteriorly (upper 40%) during the period of peak cyst output. The number of trophozoites in the small intestine and cyst excretion were found to be proportional at all stages of infection.     

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 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.     

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.     

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.     

Trypanosoma cruzi: Role of different antibody classes in protection against infection in the mouse

Immune serum obtained from mice with a chronic infection of Trypanosoma cruzi was fractionated on Sephadex G-200 or on protein ASepharose 4B. Mice were infected with a standard infective dose of T. cruzi 24 hr after injection with either IgM, IgG, IgG1, or IgG2 + IgG3 fractions. Mice were also pretreated with immune serum depleted by affinity chromatography of either IgG2a, IgG2b, or both subclasses before infection with T. cruzi. Control mice were pretreated with normal mouse serum or immune serum depleted of IgG. The parasitemia and survival of the animals were determined and used as parameters of protection. The results of these experiments demonstrated that the protective antibodies were mostly IgG2 and seem to be preferentially located in IgG2b subclass. IgM and IgG1 fractions were very little, if any, protection.     

Herpetomonas samuelpessoai: Changes in cell shape and induction of differentiation by local anesthetic

Lidocaine, a local anesthetic, induces changes in morphology and motility of Herpetomonas samuelpessoai when incubated under nonproliferative conditions. The cells become rounded and immotile. These effects are dependent on time and temperature of incubation, concentration of lidocaine, and pH. Divalent cations (Ca²⁺, Mg²⁺, Ba²⁺, and Sr²⁺) reversed the effect of lidocaine. Lidocaine also induces the formation of membrane-bound cytoplasmic vacuoles as determined by morphometry applied to electron micrographs. Lidocaine had a dose-dependent effect on the growth of H. samuelpessoai in a chemically defined medium. At concentrations which did not interfere with cell growth, it induces the transformation of promastigote into opisthomastigote via paramastigote. It is suggested that lidocaine may be used as an inductor of differentiation in H. samuelpessoai opening the possibility of obtaining the three developmental stages of this trypanosomatid.     

Trypanosoma brucei: Biochemical and morphological changes during in vitro transformation of bloodstreamto procyclic-trypomastigotes

Trypanosoma brucei brucei in whole rat blood inoculated into a semidefined medium undergoes complete morphological transformation (observed by light microscopy) in 72 hr. This reproducible system permits the biochemical and physiological study of transformation from bloodstream to procyclic trypomastigotes and mitochondrial biogenesis in these organisms. Infectivity for mice is lost after 6 days. Proline stimulates cell growth after transformation. High levels of glucose adversely affect the growth of transforming cells. Respiration during transformation is by an α-glycerophosphate oxidase although a cyanide-sensitive pathway is present after 24–48 hr but does not become fully functional with respect to procyclic trypomastigotes until 20–24 days in culture. The success of this system will permit the biochemical characterization of African trypanosomes as the development of the cytochrome system occurs.     

Theileria parva: Kinetics of infection in the lymphoid system of cattle

The kinetics of infection with Theileria parva in cattle were studied by examining the total cellularity and numbers of parasites in a range of lymphoid organs from animals killed at intervals during the course of the infection. With the dose of T. parva stabilate used, macroschizonts were initially detected in the drainage lymph node about 7 days after inoculation and death of the host resulted on Day 18–19. Associated with the initial detection of parasites, there was a marked increase in cellularity of the drainage lymph node and a more gradual and less pronounced increase in cellularity of the other lymphoid organs. From about Day 12 onward, there was a gradual decrease in the cellularity in all of the lymphoid organs, so that in animals examined in the terminal stages of the infection there was often cellular depletion. The pattern of these cellular changes was similar in groups of Boran and Friesian cattle, although both the increase in cellularity and the terminal depletion were more marked in the Friesians. Blood leukocyte counts in infected Boran started to drop as early as Day 7 of infection and by Day 14 had reached values less than 25% of normal. Quantitation of parasitic schizonts indicated that the numbers of parasites in the lymphoid organs do not increase in a simple exponential manner. Rather, there appears to be an early rapid increase in parasite numbers followed by a phase of less rapid multiplication. Because of the marked changes which occured in total cellularity of the lymphoid organs during the course of the infection, a significant discrepancy was found between the replication rate of the parasite as calculated using total numbers of parasites and that obtained using schizont index (SI). These results indicated that the use of SI, as described in previous studies, is not a reliable method of determining the replication rate of the parasite.     

Plasmodium berghei: Uptake and distribution of chloroquine in infected mouse erythrocytes

The capacity of mouse erythrocytes infected with Plasmodium berghei to accumulate chloroquine is developed with maturation of the parasites. This is shown by direct comparison of the early and mature stages, which are separated by density difference. After drug accumulation, infected cells were fractionated by saponin lysis or nitrogen decompression to study the drug distribution. Effectiveness of isolating intact parasites and host components was checked by SDS-polyacrylamide gel electrophoresis and by low leakage of parasite-specific lactate dehydrogenase used as a marker enzyme. At low external drug concentration (~10^?7M), chloroquine is principally accumulated in the parasites. However, at higher drug concentrations (~10^?5and ~10^?3M), the proportion of the drug found in the host cytosol fraction is increased. A small but significant proportion of the drug (<20%) is associated with the host cell membrane. The pellet fraction of the freed parasites, further fractionated by freeze-thaw lysis, contains a major proportion of the drug at low external concentrations. However, the pellet fraction obtained from prolonged sonication of the parasites, which contains the bulk of hemozoin pigment, carries only a small proportion of the drug. This indicates that parasite membrane components may bind most of the drug. As external chloroquine concentration is increased, the proportion of drug in the parasite supernatant increases, some or most of which is probably bound by soluble hemecontaining compounds. However, the presence of chloroquine in the parasite does not affect the partition of heme in particulate and soluble forms.     

Trypanosoma congolense: Natural and acquired resistance in the bovine

A total of 42 animals of various ages were infected with Trypanosoma congolense to investigate age resistance. Ten of eleven animals between 4 months and 1 year of age survived the infection without treatment. Two of eleven animals in the age range of 1 to 2 years also survived the infection whereas all 20 animals between 2 and 5 years of age died or needed treatment to survive. Young animals which needed no treatment to survive were refractive to challenge for at least 1 year after their last patent parasitemia. Older animals which required treatment to survive were also challenged at intervals after therapy. Three animals infected for 49 to 75 days before treatment were rechallenged 198 to 296 days later. Extensions in prepatent periods ranged from 5 to 13 days when compared to controls and the resulting infections were of a relapsing nature followed by self-cure. Effects of this disease on clinical parameters of previously infected animals were minimal. One animal infected for 196 days and rechallenged 501 days later had a prepatent period of 14 days as compared to 5 days for controls. This animal developed a brief relapsing infection followed by self-cure. Animals which were infected for periods of 41 to 77 days, received treatment, and were then rechallenged from 600 to 900 days later, showed some resistance to infection. Prepatent periods were extended from 1 to 3 days over those of control animals and although the resulting disease was severe, one of four animals self-cured without treatment. When animals which had self-cured primary challenges were rechallenged at periods up to 2 years later, they were completely refractory. When 12 animals which were presumed to be immune to syringe-passaged T. congolense were challenged by tsetse fly bite with the same strain of trypanosome, an appreciable immunity was evident. Five of twelve immune animals did not become patent while the other seven developed mild infections without severe clinical signs. All nine controls developed severe infections with eight requiring treatment to survive. When animals immune to the Trans-Mara I strain of T. congolense were challenged either by syringe or tsetse fly bite with a heterologous strain of T. congolense obtained from a different geographical area, no evidence of immunity was detected.     

Trypanosoma vivax: Courses of infection with three stabilates in inbred mouse strains

The courses of infection in inbred mouse strains were compared following infection with three Stabilates of high, intermediate, and low virulence of Trypanosoma vivax stock Zaria Y486. Mouse strains could only be shown to differ in their resistance to T. vivax infections as judged by the height of the initial parasitemia and survival times when a trypanosome population of low or intermediate virulence was used. A T. vivax population of high virulence was uniformly lethal. Comparison of lytic antibody titers between groups of resistant ($\text{C}\text{57}\text{B}\text{1}\text{6}$) and susceptible ($\text{Balb}\text{c}$) mice did not show any significant differences in titers of the surviving mice but the mice in either group which did not control the initial parasitemia had lower lytic antibody titers than those which did. A significantly larger number of $\text{Balb}\text{c}$ mice failed to control the initial infection as compared to the $\text{C}\text{57}\text{B}\text{1}\text{6}$. Treatment with cyclophosphamide did not ablate differences in susceptibility between the two strains. The use of congenic mice showed that these differences in susceptibility were not related to differences in the major histocompatibility complex between these strains.     

Plasmodium falciparum: Attenuation by irradiation

The effect of irradiation on the in vitro growth of Plasmodium falciparum was investigated. The cultured malarial parasites at selected stages of development were exposed to gamma rays and the sensitivity of each stage was determined. The stages most sensitive to irradiation were the ring forms and the early trophozoites; late trophozoites were relatively insensitive. The greatest resistance was shown when parasites were irradiated at a time of transition from the late trophozoite and schizont stages to young ring forms. The characteristics of radiosensitive variation in the parasite cycle resembled that of mammalian cells. Growth curves of parasites exposed to doses of irradiation upto 150 gray had the same slope as nonirradiated controls but parasites which were exposed to 200 gray exhibited a growth curve which was less steep than that for parasites in other groups. Less than 10 organisms survived from the 10⁶ parasites exposed to this high dose of irradiation; the possibility exists of obtaining radiation-attenuated P. falciparum.     

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.     

Plasmodium berghei: The in Vitro immune response

Following primary in vitro Stimulation by Plasmodium berghei, IgM titers by the indirect fluorescent antibody test (IFAT) were negative in in vitro reconstituted syngeneic mouse spleen cultures containing T cells and macrophages, or B cells and macrophages, or macrophages alone, but IgM titers of 1:20 were obtained from cultures containing B cells, T cells, and macrophages. IFAT IgG titers were negative for cultures with T cells and macrophages together, or macrophages alone, but rose to 1:40 with cultures containing B cells and macrophages and 1,80 with cultures of B cells, T cells, and macrophages together. After a second in vitro challenge, IgM and IgG titers were similar to the stimulated cultures from immunized mice; the IgM titer reached only 1:20 but the IgG titer rose to 1:160. Total immunoglobulin was higher in the cultures from immunized mice than from in vitro primed cultures.     

Plasmodium falciparum: Drug sensitivity in vitro of isolates before and after adaptation to continuous culture

Fifteen strains of Plasmodium falciparum have been cultivated since 1979 using the Trager and Jensen method of continuous culture on isolates from malaria patients. One hundred and two drug sensitivity studies have been carried out on these strains using a semimicro test. Three isolates, initially resistant to chloroquine, adapted rapidly to in vitro cultivation and maintained their high level of resistance (ED₅₀ above 660 nM). Eleven isolates, initially chloroquine sensitive (ED₅₀ under 90 nM) became resistant to this drug (ED₅₀ = 190 to 1950 nM) after the 2–15 weeks required for their adaptation to continuous culture. The resistance of these strains never decreased during the following 15 months of continuous culture. The sensitivity to quinine varied initially from one strain to another (ED₅₀= 160 to 660 nM) and fluctuated during cultivation in the ratio of 1, 3.5 for a given strain. The sensitivity of mefloquine remained high for all strains (ED₅₀ under 150 nM) but one (ED₅₀ = 560 nM). These results suggest that there might be a relationship between in vitro adaptation to culture of P. falciparum by the Trager-Jensen method and a chloroquine-resistant characteristic of the strain. There is the possibility of the emergence of a drug-resistant subpopulation or of changes in the metabolic pathways.     

Entamoeba histolytica and Giardia lamblia: Growth responses to reducing agents

The growth responses of Entamoeba histolytica strains HM-1:IMSS and HK-9 to a variety of reducing agents were tested for one subculture in TYI-S-33 medium, prepared with no cysteine or ascorbic acid. Amoebae did not grow in this medium. Addition of l-ascorbic acid, d- or l-cysteine, or l-cystine each permitted the maximum growth observed. Dithiothreitol supported 68% maximum growth of HK-9 amoebae, but only 12% of HM-1. In contrast, growth of both strains was greatly diminished (0–13% growth) with 11 other compounds tested including glutathione, thiomalic acid, thioglycolic acid, and methionine. The growth responses of Giardia lamblia were similarly tested in TYI-S-33, as well as in TP-S-1 media. If l-cysteine was omitted from either medium, trophozoites did not grow, and eventually lysed. In TYI-S-33 medium, the requirement for l-cysteine was specific, whereas in TP-S-1 medium, other sulfhydryl compounds were partially effective and lower concentrations of l-cysteine satisfied the requirement. Ascorbic acid or l-cystine alone was totally ineffective; however, in combination, 30 to 60% of maximum growth was achieved. Once added to either medium, cysteine was rapidly oxidized. Amino acid analysis of the growth media revealed that the broth components of TP-S-1 medium contained 2.8 mM and TYI-S-33 broth 2.1 mM endogenous levels of cysteine (or half-cystine), with an additional 3 mM contributed by 10% serum.