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.     

Eimeria tenella: Parasite-Specific Incorporation of 3H-Uracil as a Quantitative Measure of Intracellular Development1

ABSTRACT. An assay has been developed using parasite-specific incorporation of ³H-uracil to assess the intracellular growth of Eimeria tenella in vitro. As shown by both scintillation counts and autoradiography, ³H-uracil was incorporated specifically into intracellular parasites from the onset of infection and continued throughout development of the first generation schizonts. Mature schizonts and first generation merozoites did not continue to incorporate additional ³H-uracil, indicating that RNA synthesis had halted in these stages. Based on these findings, a semi-automated microscale uracil incorporation assay was developed to determine parasite viability. This method should be useful for biochemical studies with intracellular parasites and for screening compounds for anticoccidial activity. The ease, rapidity, and quantitative nature of this assay contrasts favorably with standard morphometric approaches of determining parasite development. In addition, parallel studies using host cell incorporation of ³H-uridine have been introduced as a method of determining whether antiparasitic activity is direct or indirect in relation to effects on the host cell.     

Anticoccidial drugs: Effects on infectivity and survival intracellularly of Eimeria tenella sporozoites

The effects of various anticoccidial drugs on extracellular and intracellular sporozoites were studied in cell culture and in chickens. Treatment of freshly excysted, extracellular sporozoites of Eimeria tenella for 18 hr with monensin, decoquinate, or robenidine at 100 ppm had no effect on oocyst production 7–10 days after the sporozoites were rinsed free of drugs and fed to chickens. Treatment of cultures of E. tenella in chick kidney cell monolayers with monensin (0.001 μg/ml), decoquinate (0.01 μg/ml), zoalene (20.0 μg/ml), or robenidine (0.01 μg/ml) had no effect on intracellular sporozoites at 4 hr following introduction of sporozoites and drugs into the culture. A significant reduction of intracellular parasites occurred at 24 hr in the cultures treated with monensin or zoalene. Remaining intracellular sporozoites in monensin-treated cultures were morphologically abnormal or degenerate, while sporozoites in other cultures appeared normal. The number and condition of sporozoites in the nontreated cultures were unchanged at 24 hr postinoculation. These results indicate that sporozoites undergo changes subsequent to penetration of host cells that render them susceptible to drug action.     

Eimeria tenella: synergistic interaction of sulfaquinoxaline and t-butylaminoethanol in the chicken.

A significant (P < 0.001) synergistic interaction between sulfaquinoxaline and t-butylaminoethanol was demonstrated against a sulfaquinoxaline-resistant strain of Eimeria tenella in chickens. The t-butylaminoethanol is a dimethylaminoethanol- and choline-reversible anticoccidial whereas sulfaquinoxaline is a classic PABA-reversible anticoccidial.     

Endogenous Cycle of the Swine Coccidium Eimeria debliecki Douwes, 1921*

SYNOPSIS. A pure strain of Eimeria debliecki (University of Illinois strain A) established from a single oocyst was used to determine the endogenous cycle. Young parasite-free pigs 2 weeks to 3 months old were used throughout the study. The endogenous cycle was found to take place in the small intestine where the parasites were located in the distal portion of the striated simple columnar epithelial cells of the villi. The first generation schizonts were found in only the jejunum (15% of small intestine). The second generation schizonts and gametes occurred in the jejunum and ileum (70% of small intestine), a slight posterior progression occurring with each stage. The entire cycle required 6.5 days. The schizogonous cycle comprised 2 generations. The first generation schizonts required 2.5 days to reach maturity, measured 8-12 μ, contained 16 merozoites measuring 12-15 μ and had a polar residual mass. The second generation schizonts required 2 days to reach maturity, measured 13-16 μ, contained 32 rotund merozoites measuring 6–8 μ, and had only a few granules of residual material. Gametogony took place in 1.5 days. The macrogametes measured 12-16 μ, and the microgametocytes measured 9-14 μ with microgametes measuring 5–6 μ.     

Effect of artemisinin on oocyst wall formation and sporulation during Eimeria tenella infection

The anticoccidial effect of a product extracted from the natural herb Artemisia annua, artemisinin, which has a potential use as a dietary supplement, has been studied. Commercial artemisinin was administered at 10 and 17 ppm in food and tested against infection with Eimeria tenella. A battery trial to quantify the effect of artemisinin on the reproductive and infective capabilities of E. tenella was carried out. For that purpose flow cytometry was combined with electron microscopy and immunofluorescence techniques in order to study the effect of artemisinin on E. tenella gametogenesis. Significantly reduced oocyst output and lesion scores were found in chickens treated with artemisinin. In addition, evidence to support a lower oocyst sporulation rate was obtained. Though the ultrastructural studies showed normal development of gametogenesis in artemisinin-treated chickens, the oocyst wall formation was significantly altered. This resulted in both death of developing oocysts and reduced sporulation rate. Immunofluorescent studies provided evidence that treatment with artemisinin inhibited sarcoplasmic–endoplasmic reticulum calcium ATPase (SERCA) expression in macrogametes. According to these findings, artemisinin has a deleterious effect on fertilized macrogametes (early zygotes) by inhibiting SERCA. The altered secretion of the wall-forming bodies may be the result of Ca²⁺-dependent ATPase impaired activity which, in turn, is the result of SERCA inhibition.     

Pre-Erythrocytic Development and Associated Host Responses to Haemoproteus meleagridis (Haemosporina: Haemoproteidae) in Experimentally Infected Domestic Turkeys12

ABSTRACT. Two generations of pre-erythrocytic schizogony occurred in skeletal and cardiac muscle of domestic turkeys infected with sporozoites of Haemoproteus meleagridis. First generation schizonts reached maturity approximately five days post-inoculation (DPI) and developed in capillary endothelial cells and myofibroblasts. The schizonts ranged from 12 to 20 μm in diameter and produced long (5–6 μm), slender merozoites. Early second generation schizonts were first detected in capillary endothelial cells between 5 and 8 DPI. They were cylindrical and ranged in size from 5 to 8 μm in diameter and up to 28 μm in length. Second generation schizonts which reached maturity by 17 DPI were surrounded by a thick, hyaline wall and were packed with numerous spherical merozoites less than 1 μm in diameter. Mature megaloschizonts were fusiform, ranged from 30 to 113 μm in diameter, and extended as much as 465 μm along the long axis of muscle fibers. Merozoites developed as buds from cytomeres that formed between 8 and 14 DPI. Infected turkeys developed a moderate to severe myositis within 5 DPI and were lame in one or both legs. The myositis was associated with the necrosis of scattered groups of muscle fibers. Muscle fibers surrounding mature megaloschizonts were swollen and hyaline. Megaloschizonts were surrounded occasionally by fibroblasts and infiltrates of mononuclear cells. The morphology and site of development of mature megaloschizonts of Haemoproteus meleagridis are contrasted with those of other avian haemosporidians.     

Development of cross-protective Eimeria-vectored vaccines based on apical membrane antigens

Recently, the availability of protocols supporting genetic complementation of Eimeria has raised the prospect of generating transgenic parasite lines which can function as vaccine vectors, expressing and delivering heterologous proteins. Complementation with sequences encoding immunoprotective antigens from other Eimeria spp. offers an opportunity to reduce the complexity of species/strains in anticoccidial vaccines. Herein, we characterise and evaluate EtAMA1 and EtAMA2, two members of the apical membrane antigen (AMA) family of parasite surface proteins from Eimeria tenella. Both proteins are stage-regulated, and the sporozoite-specific EtAMA1 is effective at inducing partial protection against homologous challenge with E. tenella when used as a recombinant protein vaccine, whereas the merozoite-specific EtAMA2 is not. In order to test the ability of transgenic parasites to confer heterologous protection, E. tenella parasites were complemented with EmAMA1, the sporozoite-specific orthologue of EtAMA1 from E. maxima, coupled with different delivery signals to modify its trafficking and improve antigen exposure to the host immune system. Vaccination of chickens using these transgenic parasites conferred partial protection against E. maxima challenge, with levels of efficacy comparable to those obtained using recombinant protein or DNA vaccines. In the present work we provide evidence for the first known time of the ability of transgenic Eimeria to induce cross protection against different Eimeria spp. Genetically complemented Eimeria provide a powerful tool to streamline the complex multi-valent anticoccidial vaccine formulations that are currently available in the market by generating parasite lines expressing vaccine targets from multiple eimerian species.     

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.     

Induction of host nuclear DNA synthesis in coccidia-infected chicken intestinal cells

When Eimeria maxima (gamonts) infects villus epithelial cells of the chicken duodenum there is extensive cellular enlargement with no alteration in nuclear size. Feulgen DNA microspectrophotometric measurements indicated that the infected host-cell nucleus contains the same amount of DNA as an uninfected cell nucleus. Evidence is presented to indicate that second generation schizonts of E. necatrix develop in crypt epithelial cells that are displaced/migrate into the lamina propria. The developing parasite causes cellular and nuclear hypertrophy in these cells as does E. tenella in cecal cells of the chicken. In these two cases nuclear enlargement is accompanied by induced rounds of DNA synthesis in the host-cell. Analyses indicated that the DNA content of enlarged nuclei does not fall into classes that correspond to a geometric series 2:4:6:8:16: etc. times the DNA content of a 2C equivalent, and that nuclear size and DNA content in infected cells are not significantly correlated. Autoradiographic studies on E. necatrix infected chicks administered ³H-thymidine show that DNA synthesis takes place in the nuclei of cells containing all developing stages but not mature schizonts, and that this synthesis is not a continuous process. The data suggest that intestinal cells that are capable of undergoing cell division and therefore additional rounds of DNA synthesis, can be induced by coccidial infection in the absence of concomitant cell division.     

Cytokinin-agonistic and antagonistic activities of 4-substituted-2-methylpyrrolo[2,3-d] pyrimidines, 7-deaza analogs of cytokinin-active adenine derivatives

Sixteen 4-substituted-2-methylpyrrolo[2,3-d]pyrimidines were tested for their activities as cytokinin agonists and antagonists in three bioassay systems. With the systematic variation of the 4-substituent, the activity changed consecutively from agonistic to antagonistic in the tobacco callus test, and this agonist-antagonist relationship was analysed by a steric substituent parameter as a common quantitative measure. Two compounds were found to possess bud-forming activity in the absence of added cytokinins. Eight compounds promoted betacyanin synthesis'in Amaranthus and 4 promoted germination in lettuce seeds. The 4-hydroxyethyl derivative, which was inactive in the tobacco test, suppressed betacyanin synthesis at 100 μM against 100 μM 6-(3-methyl-2-butenylamino)- purine. The 4-phenyl derivative showed antagonist activity in the tobacco test, little activity in the Amaranthus bio- assay and significant agonist activity in the lettuce seed germination. The 4-cyclobutyl derivative exerted the strongest anticytokinin activity (I₅₀ at 0.06 μM against 0.05 μM kinetin) in the tobacco callus assay, while little activity was observed in the other two assay systems.     

Trypanocidal properties, structure-activity relationship and computational studies of quinoxaline 1,4-di-N-oxide derivatives

Pyrazole and propenone quinoxaline derivatives were tested against intracellular forms of Leishmania peruviana and Trypanosoma cruzi. Both series were tested for toxicity against proliferative and non-proliferative cells. The pyrazole quinoxaline series was quite inactive against T. cruzi; however, the compound 2,6-dimethyl-3-f-quinoxaline 1,4-dioxide was found to inhibit 50% of Leishmania growth at 8.9 μM, with no impact against proliferative kidney cells and with low toxicity against THP-1 cells and murine macrophages. The compounds belonging to the propenone quinoxaline series were moderately active against T. cruzi. Among these compounds, two were particularly interesting, (2E)-1-(7-fluoro-3-methyl-quinoxalin-2-yl)-3-(3,4,5-trimethoxy-phenyl)-propenone and (2E)-3-(3,4,5-trimethoxy-phenyl)-1-(3,6,7-trimethyl-quinoxalin-2-yl)-propenone. The former possessed selective activity against proliferative cells (cancer and parasites) and was inactive against murine peritoneal macrophages; the latter was active against Leishmania and inactive against the other tested cells. Furthermore, insilico studies showed that both series respected Lipinski’s rules and that they confirmed a linear correlation between trypanocidal activities and LogP. Docking studies revealed that compounds of the second series could interact with the poly (ADP-ribose) polymerase protein of Trypanosoma cruzi.     

Nosema whitei, a microsporidan pathogen of some species of Tribolium : I. Morphology,life cycle,and generation time

The morphology of Nosema whitei is described from 4 host beetles, Tribolium castaneum, T. confusum, T. anaphe, and Oryzaephilus surinamensis. The effect of host species on the sizes of the various stages was small. The predominant schizogonic stages were mononuclear (26%) and binuclear (73%) although schizonts with up to 5 nuclei were seen. In stained preparations the schizonts were approximately 2.7–7.0 μ in diameter. The sporonts, which do not divide, were elongate (5.6 × 3.1 μ), and had 1 or 2 nuclei. Both the sporoblast (4.2 × 2.2 μ) and the spores (3.5 × 2.0 μ) were binucleate. Fresh spores averaged 4.6 × 2.9 μ. The polar filament length ranged from 75 to 135 μ (mean = 112 μ). The only tissue found infected was the fat body. Host species, dose, and temperature were all found to affect the generation time, which ranged from 8 to 17 days.     

Characterization and bioactivities of M. arvensis,V. officinalis and P. glabrum: In-silico modeling of V. officinalis as a potential drug source

In current study the pharmaceutically active herbs was used against coccidiosis, caused by a protozoan: Eimeria, lead to $ 3 billion loss annually. The aqueous and methanolic extracts of whole plants were applied in-vitro to assess sporulation inhibition (spi) assay and calculated the inhibitory concentration (IC₅₀). For in-vivo study 9 groups of 14 day old broiler chicks were infected with Eimeria tenella and three groups were treated different concentrations of methanolic extracts of Verbena officinalis and Polygonum glabrum post infection. The mean weight gain, oocyst count, diarrhea, biochemical tests, hematology, and histopathology of all groups were analyzed. The herbs were characterized by antioxidant assay, phytochemical screening, Fourier transmission and infrared (FT-IR), Ultra Violet-visible (UV–Vis) spectroscopy and Gas chromatography and mass spectroscopy (GC–MS). The GC–MS identified phyto-compounds of V. officinalis were docked with S-Adenosyl methionine (SAM) synthetase. The in-vitro study revealed that V. officinalis and P. glabrum have minimum IC₅₀ of 0.14 and 12 mg/ml respectively. The in-vivo experiment showed that V. officinalis had significantly high anticoccidial potential with significant hematological profile like drug treated controls. The histology of treated chicks also showed recovery in the studied tissues. The antioxidant assay showed that V. officinalis have 4.19U/mg Superoxide dismutase (SOD) and 33.96 µM/mg Glutathione (GSH) quantities. The chemical characterization confirmed the presence of large number of organic compounds, however Flavonoids found only in V. officinalis, which suggests the anticoccidial potential of V. officinalis because flavonoids as antagonist of thiamine (Prinzo, 1999), because it promotes the carbohydrate synthesis required. Strychane, 1-acetyl-20a-hydroxy-16-methylene has best binding of with target protein with lowest binding score (-6.4 Kcal/mol), suggests its anticoccidial potential in poultry.     

Digestive and metabolic utilization of lauric,myristic and stearic acid in cows,and associated effects on milk fat quality

A study was carried out to examine the effect of dietary supplementation of oregano essential oil on performance of broiler chickens experimentally infected with Eimeria tenella at 14 days of age. A total of 120 day-old Cobb-500 chicks separated into 4 equal groups with three replicates each, were used in this study. Two groups, one infected with 5·10⁴ sporulated oocysts of E. tenella and the other not, were given a basal diet and served as controls. The other two groups also infected with E. tenella were administered diets supplemented with oregano essential oil at a level of 300 mg/kg, or with the anticoccidial lasalocid at 75 mg/kg. Following this infection, survival rate, bloody diarrhoea and oocysts excretion as well as lesion score were determined. Throughout the experimental period of 42 days, body weight gain and feed intake were recorded weekly, and feed conversion ratios were calculated. Two weeks after the infection with E. tenella supplementation with dietary oregano oil resulted in body weight gains and feed conversion ratios not differing from the non-infected group, but higher than those of the infected control group and lower than those of the lasalocid group. These parameters correspond with the extent of bloody diarrhoea, survival rate, lesion score and oocyst numbers and indicated that oregano essential oil exerted an anticoccidial effect against E. tenella, which was, however, lower than that exhibited by lasalocid.     

Potentiation of ionophorous anticoccidials with dihydroquinolines: Battery trials against Eimeria tenella in chickens

Seven battery tests, employing a total of 1344 Hybro cockerels, were conducted in order to study the interaction between ionophorous anticoccidials and a new dihydroquinoline-type antioxidant known as duokvin. A significant, non-selective, toxic interaction was established, resulting in growth depression and improved anticoccidial efficacy against Eimeria tenella and E. mitis in these chickens. The duokvin itself showed no appreciable activity against the coccidia. The adverse effects of the interaction were eliminated, and the anticoccidial efficacy of the approved levels of ionophores was maintained, when the dietary levels of monovalent cation ionophorous monensin, salinomycin and narasin were reduced to approximately 12% in the presence of 120p.p.m. duokvin. No adverse effects on the growth of chickens appeared in the combination with maduramicin, yet the enhancement of anticoccidial activity allowed an approx. 50% reduction of this ionophore as well.     

Cytokinin-induced shoot formation

Zeatin, (±)-dihydrozeatin and optically active cytokinins (asymmetric carbon α to the exocyclic nitrogen) were tested for their ability to induce development of shoots in tobacco callus. Zeatin and dihydrozeatin were equally active. The levorotatory compounds tested were active in inducing shoot formation but the corresponding dextrorotatory compounds were inactive at all concentrations tested. These findings suggest that the group attached to the N⁶ position of cytokinins binds to a receptor site to bring about organ formation.     

Anticoccidial Activity of Berberine against Eimeria-Infected Chickens

Avian coccidiosis has a major economic impact on the poultry industry, it is caused by 7 species of Eimeria, and has been primarily controlled using chemotherapeutic agents. Due to the emergence of drug-resistant strains, alternative control strategies are needed. We assessed anticoccidial effects of berberine-based diets in broiler chickens following oral infection with 5 Eimeria species (E. acervulina, E. maxima, E. tenella, E. mitis, and E. praecox). When 0.2% berberine, a concentration that does not affect weight gain, was added to the diet, the 4 groups infected with E. acervulina, E. tenella, E. mitis, or E. praecox showed significant reductions in fecal oocyst shedding (P<0.05) compared to their respective infected and untreated controls. In chickens treated 0.5% berberine instead of 0.2% and infected with E. maxima, fecal oocyst production was significantly reduced, but body weight deceased, indicating that berberine treatment was not useful for E. maxima infection. Taken together, these results illustrate the applicability of berberine for prophylactic use to control most Eimeria infections except E. maxima. Further studies on the mechanisms underlying the differences in anticoccidial susceptibility to berberine, particularly E. maxima, are remained.     

In vivo anticoccidial activity of berberine [18, 5,6-dihydro-9,10-dimethoxybenzo(g)-1,3-benzodioxolo(5,6-a) quinolizinium] – An isoquinoline alkaloid present in the root bark of Berberis lycium

Coccidiosis, caused by various Eimeria species, is a major parasitic disease in chicken. However the increasing resistance of these parasites to currently used anticoccidial drugs has stimulated the search for new methods of control. As part of this effort we investigated the root bark of Berberis lycium (barberry) as a potential source of compounds with anticoccidial activity. In the present study anticoccidial activity of different solvent extracts of the root bark of B. lycium and berberine was evaluated in vivo using broiler chicken. Results of the study demonstrated equipotent efficacy of pure berberine in comparison to that of standard drug amprolium on the basis of reduction in coccidian oocyst output, body weight gain of chicken and feed conversion ratio. Among the extracts crude methanolic extract showed highest anticoccidial activity tested at 300 mg/kg body weight which could be due to the presence of alcohol-soluble active ingredients in root bark of B. lycium. Toxicological studies revealed that B. lycium extracts as well as berberine were not lethal up to dosage of 2000 mg/kg body weight. LD⁵⁰ was not determined as mortalities were not recorded in any of the five groups of chicken. From the present study it can be concluded that root bark of B. lycium has the immense potential to contribute to the control of coccidian parasites of chicken. Our results corroborate the use of berberine for treatment of severe diarrhoea, amoebiasis and intestinal infections and could justify its use in folk medicine for treatment of haemorrhagic dysentery.     

Eimeria tenella: specific reversal of t-butylaminoethanol toxicity for parasite and host by choline and dimethylaminoethanol.

t-Butylaminoethanol is an anticoccidial compound that is related structurally to the metabolically active substances, dimethylaminoethanol, and choline. Toxic effects of t-butylaminoethanol for chickens and Eimeria tenella are specifically overcome by feeding sufficient amounts of dimethylaminoethanol or choline. Dietary concentrations of the two above metabolites required to totally overcome toxic effiects of t-butylaminoethanol were determined and are expressed as the reversal ratio, inhibitor (t-butylamino-ethanol): metabolite. The inhibitor:choline ratio for total reversal of toxic effects of the inhibitor in chickens is approximately 1:10 over a concentration range of inhibitor from 0.019 to 0.05%. The inhibitor:choline ratio for reversal of antiparasitic effects is approximately 1:200 with a concentration of 0.01% inhibitor. The inhibitor:Dimethylaminoethanol ratio for reversal of toxic effects of the inhibitor in the chicken is approximately 1:7 with a concentration of 0.015% inhibitor. The inhibitor:dimethylaminoethanol ratio for reversal of antiparasitic effects is approxmately 1:20 wth a concentration of 0.01% inhibitor.