Eimeria tenella: anticoccidial action of drugs in birds with surgically closed ceca.

Surgical ligation of chick ceca was used to study the role of absorption and extraintestinal transport in the action of anticoccidial drugs. The administration of drugs in the feed was started after ligation of one of the paried ceca. Birds were inoculated orally with oocysts of Eimeria tenella before cecal ligation or were given bilateral cecal injections of sporozoites after ligation. Cecal lesions caused by the coccidia were evaluated and compared on day 6 postinoculation. Lesions in ligated and unligated ceca were reduced by feeding robenidine (33 ppm), arprinocid (70 ppm), zoalene (125 ppm), aklomide (250 ppm), clopidol (125 ppm), nicarbazin (125 ppm), monensin (120 ppm), salinomycin (60 ppm), and lasalocid (75 ppm). The lesions were more severe in the ligated cecum than in the intact cecum, whether in nonmedicated or medicated birds, but the differences were statistically significant only upon treatment with amprolium, aklomide, robenidine, and clopidol. Generally, however, all drugs except amprolium, significantly reduced the lesions in the ligated cecum in comparison with the control, nonmedicated ligated cecum. Therefore, we concluded that the systemic absorption of most anticoccidial drugs contributes significantly to their efficacy against coccidia in the intestinal mucosa.     

Anti-coccidial activity of 2-picoline, 6-amino-4-nitro-, 1-oxide

An investigational drug (2-picoline, 6-amino-4-nitro-, 1-oxide) was evaluated to characterize the anti-coccidial spectrum of the compound. Two concentrations of the drug (125 and 250 ppm) were evaluated for bioactivity; weight gain, survival, dropping, and lesion scores were the response variables utilized to ascertain activity. The activities of the picoline derivative were compared with monensin, maduramicin, and a narasin/nicarbazin (1:1) combination. The investigational drug had significant activity against Eimeria tenella and Eimeria necatrix, and the 250-ppm level was significantly more active than 125 ppm. At 250 ppm, the E. tenella activity of the picoline derivative was comparable to both monensin (120 ppm) and the 50-ppm narasin/nicarbazin combination, significantly less effective than maduramicin (6 ppm), and significantly more efficacious than 30 ppm narasin/nicarbazin. At the same level (250 ppm), the picoline derivative had significantly less E. necatrix activity than monensin (120 ppm), maduramicin (6 ppm), and narasin/nicarbazin (50 ppm), and significantly greater activity than 30 ppm narasin/nicarbazin. At best, only extremely weak Eimeria acervulina, Eimeria brunetti, and Eimeria maxima activities were noted with the investigational drug; higher concentrations of the picoline derivative may achieve greater anti-coccidial activity against these species. The efficacy of narasin/nicarbazin compared favorably with monensin and maduramicin; the 50-ppm level of the combination appeared significantly more efficacious than 30-ppm.     

Characterization in vitro and in vivo of resistance to ionophores in a strain of Eimeria tenella.

A field isolate of Eimeria tenella (FS139) was propagated several times in chickens medicated with 200 ppm of dietary monensin. In a laboratory test with 2-wk-old-chickens, the strain was resistant to monensin, salinomycin, and lasalocid given at double use level and was resistant to narasin and maduramicin at the normal use level. In comparison, a laboratory strain (WIS) was controlled by the normal use level of each product. When free WIS sporozoites were treated in vitro with 1.0 microgram/ml of monensin for 0.5 or 4.0 hr at 41 C and inoculated into primary cultures of chicken kidney cells the invasion was reduced by 35.6% or 96.3%, but invasion of FS139 sporozoites was increased by 18.5% by 0.5 hr treatment and was about the same as controls after 2 hr of treatment. Few sporozoites from the WIS strain developed into schizonts, but numerous sporozoites from the FS139 strain developed into normal first and second generation schizonts. The structure of free WIS sporozoites was distorted after 3 hr of treatment with 2.5 micrograms/ml of monensin at 41 C, as observed by light and scanning electron microscopy, whereas there was no change in structure of most treated FS139 sporozoites.     

Influence of monensin on cation influx and Na+ -K+ -ATPase activity of Eimeria tenella sporozoites in vitro

The experiments were conducted to determine intrasporozoite Na+/K+ concentrations (by AAS) and membrane-bound Na+ -K+ -ATPase activity (measured by UV-VIS with a Na+ -K+ -ATPase Detection Kit) of Eimeria tenella sporozoites of the sensitive line (i.e., the parent line, coded as OS) and 2 resistant lines, derived from the parent line (coded as OR125 and OR200), with and without in vitro exposure to monensin. These parameters for OR125 and OR200 were significantly lower than those for OS. In vitro exposure to monensin increased intrasporozoite Na+/K+ concentrations and Na+ -K+ -ATPase activity, but the stimulation on OS was significantly higher than those on OR125 and OR200, indicating that monensin had less effect on resistant parasites. The results of this study suggest that altered biochemical or physiological properties, or both, in the membranes of E. tenella might be related to a reduced sensitivity to monensin.     

Studies on short-term application of drugs to Eimeria tenella in tissue culture.

The anticoccidial action of 6 drugs was studied by treating cell cultures with drug for 24 h after inoculation of the cultures with sporozoites of Eimeria tenella. A monitoring technique was used to confirm that the drug was being removed. Only monensin was shown to be coccidiocidal. Amprolium, lasalocid, methyl benzoquate, nicarbazin and robenidine were coccidiostatic at their minimum effective doses. Misleading coccidiocidal effects were observed when higher concentrations of methyl benzoquate, nicarbazin or robenidene were used.     

Interaction of T-2 fusariotoxin with anticoccidial efficacy of lasalocid in chickens.

Two battery tests were carried out to reveal whether or not there is an interaction between T-2 fusariotoxin and lasalocid, similar to that known with some ionophorous anticoccidials. In the first experiment, an excessive dose level of toxin (6 ppm) significantly reduced the anticoccidial efficacy of lasalocid at 75 ppm and almost completely eliminated that at 37.5 ppm in cockerels which had been infected with oocysts of Eimeria tenella and E. mitis. In the second experiment, the same dose of lasalocid was tested in combination with 0.5-1.25 ppm levels of toxin, i.e. levels occurring in the field, and all but the lowest level (0.5 ppm) significantly reduced the anticoccidial efficacy of lasalocid. Thus, in cases of outbreaks of coccidiosis, there is need to keep in mind such interaction between anticoccidials and mycotoxins.     

Factors influencing the assessment of anticoccidial activity in cell culture

A comparative study has been made of the factors influencing the assessment of anticoccidial potency in vitro against Eimeria tenella using established anticoccidials and arprinocid and some of its analogues. Drugs whose potency depended upon medium composition were amprolium, lasalocid and halofuginone. There was a difference in strain sensitivity with robenidine. Host cell type had an important effect on potency of monensin, decoquinate, arprinocid and its analogues. Arprinocid was active in chick liver cell systems but totally inactive in chick kidney cell systems, although its N-oxide was active in both cell types. Arprinocid-containing medium, conditioned by supporting the growth of chick embryo liver cell cultures, had an anticoccidial effect on E. tenella growing in chick kidney cells. It is deduced that the anticoccidial activity of arprinocid in the chick is due to a metabolite.     

Expression of flotillin-1 on Eimeria tenella sporozoites and its role in host cell invasion

Lipid rafts are detergent-resistant, liquid-ordered microdomains in plasma membranes that are enriched in cholesterol and sphingolipids and involved in intracellular signal transduction, membrane trafficking, and molecular sorting. In this study, we investigated the possibility that lipid rafts on Eimeria tenella sporozoites may act as platforms for host cell invasion. Flotillin-1, a resident protein of lipid rafts, was identified on E. tenella sporozoites and was prominently expressed at the apex of the cells, a region mediating host cell invasion. Pretreatment of sporozoites with antibody against flotillin-1 blocked parasite invasion. Furthermore, the anticoccidial drug, monensin, disrupted the localization of flotillin-1 within raft structures resulting in loss of invasion. We conclude that Eimeria sporozoites utilize lipid rafts containing flotillin-1 for internalization into host cells.     

The site of action of the anticoccidial salinomycin (Coxistac).

The anticoccidial salinomycin has a cidal effect against chicken coccidia. Restricted and unrestricted medication studies and histopathological examinations of chicks infected with Eimeria acervulina, E. maxima, or E. tenella showed that parasites were destroyed within host cells during asexual development. Most sporozoites failed to become trophozoites and were destroyed 30--72 hr after ingestion of oocysts. The drug also affected schizonts during initial nuclear replication by either destroying or significantly delaying their maturation. Parasites affected by the drug were distorted grossly. Drug action against gametogony was not observed histologically, but when medication was restricted to this period of the life cycle, subsequent oocyst shedding of all 3 species was reduced by 20--70% compared to unmedicated controls. When drug was provided during the entire parasite life cycle, activity against asexual stages was so complete that only a limited number of parasites survived to form gamonts, and oocyst shedding was reduced by 80--90% relative to controls. As with other ionophores, salinomycin had no effect upon rate of oocyst sporulation.     

Eimeria tenella, E. necatrix, E. acervulina, E. maxima, and E. brunetti: potent anticoccidial activity of an uridine analog, 1-(beta-D-ribofuranosyl)-2(1H)-pyrazinone 4-oxide

The anticoccidial activity of an uridine analog, 1-(beta-D-ribofuranosyl)-2(1H)-pyrazinone 4-oxide (emimycin riboside), against five species of chicken Eimeria was tested individually in battery experiments. With 16 ppm of the compound in feed, marked anticoccidial activity was obtained against Eimeria tenella, E. necatrix, E. acervulina, E. maxima, and E. brunetti. The last named species was more drug-sensitive than the others--dietary levels of at least 8 ppm of the drug exhibited good protection and eliminated practically all clinical signs. The battery tests with delayed and restricted medications showed that emimycin riboside affected the development of parasites in first and second generation schizogony of the life cycle of E. tenella.     

Methane Production by Fermentor Cultures Acclimated to Waste from Cattle Fed Monensin, Lasalocid, Salinomycin, or Avoparcin

The ability of microorganisms to ferment waste from cattle fed monensin, lasalocid, or salinomycin to methane was determined. Continuously mixed anaerobic fermentors with 3-liter working volumes at 55°C were used; fermentors were fed once per day. Initially, all fermentors were fed waste without antibiotics at 6% volatile solids (VSs, organic matter) and a 20-day retention time (RT) for 60 days. Waste from animals fed monensin, lasalocid, or salinomycin at 29, 20, and 16.5 mg per kg of feed, respectively, was added to duplicate fermentors at the above VSs, and RT. Avoparcin (5 to 45 mg/liter) was not fed to animals but was added directly to duplicate fermentors. Lasalocid and salinomycin had minimal effects on the rate of methane production at RTs of 20 days and later at 6.5 days. Avoparcin caused an increase in organic acids from 599 to 1,672 mg/liter (as acetate) after 4 weeks, but by 6 weeks, acid concentrations declined and the rate of methane production was similar to controls at a 6.5-day RT. The monensin fermentors stopped producing methane 3 weeks after antibiotic addition. However, after a 6-month acclimation period, the microorganisms apparently adapted, and methane production rates of 1.65 and 2.51 liters per liter of fermentor volume per day were obtained with 6% VSs, and RTs of 10 and 6.5 days, respectively. This compares with 1.78 and 2.62 liters/liter per day for controls (P > 0.05). All fermentors that were fed waste containing antibiotics had lower pH values and ammonia and alkalinity concentrations, suggesting less buffering capacity and protein catabolism than in controls. Acclimation results obtained with fermentors at 35°C were similar to those for fermentors at 55°C. These studies indicate that waste from cattle fed these selected growth-promoting antibiotics can be thermophilically fermented to methane at RTs of 6.5 days or longer and VS concentrations of 6%, at rates comparable to waste without antibiotics.     

Characterization of monoclonal antibodies that recognize the Eimeria tenella microneme protein MIC2

The apicomplexan pathogens of Eimeria cause coccidiosis, an intestinal disease of chickens, which has a major economic impact on the poultry industry. Members of the Apicomplexa share an assortment of unique secretory organelles (rhoptries, micronemes and dense granules) that mediate invasion of host cells and formation and modification of the parasitophorous vacuole. Among these, microneme protein 2 from Eimeria tenella(EtMIC2) has a putative function in parasite adhesion to the host cell to initiate the invasion process. To investigate the role of EtMIC2 in host parasite interactions, the production and characterization of 12 monoclonal antibodies (mabs) produced against recombinant EtMIC2 proteins is described. All mabs reacted with molecules belonging to the apical complex of sporozoites and merozoites of E. tenella, E. acervulina and E. maxima in an immunofluorescence assay. By Western blot analysis, the mabs identified a developmentally regulated protein of 42 kDa corresponding to EtMIC 2 and cross-reacted with proteins in developmental stages of E. acervulina. Collectively, these mabs are useful tools for the detailed investigation of the characterization of EtMIC2 related proteins in Eimeria species.     

Comparative study on the effects of salinomycin,monensin and meso-2,3-dimercaptosuccinic acid on the concentrations of lead,calcium, copper,iron and zinc in lungs and heart in lead-exposed mice

Background and aimEnvironmental lead (Pb) exposure damages the lungs and is a risk factor for death from cardiovascular disease. Pb induces toxicity by a mechanism, which involves alteration of the essential elements homeostasis. In this study we compare the effects of salinomycin (Sal), monensin (Mon) and meso-2,3-dimercaptosuccinic acid (DMSA) on the concentrations of lead (Pb), calcium (Ca), copper (Cu), iron (Fe) and zinc (Zn) in the lungs and heart of lead-exposed mice.MethodsSixty days old male ICR mice were divided into five groups: control (Ctrl) – untreated mice obtained distilled water for 28 days; Pb-intoxicated group (Pb) – exposed to 80 mg/kg body weight (BW) Pb(NO₃)₂ during the first 14 days of the experimental protocol; DMSA-treated (Pb + DMSA) – Pb-exposed mice, subjected to treatment with an average daily dose of 20 mg/kg BW DMSA for two weeks; Monensin-treated (Pb + Mon) – Pb-exposed mice, obtained an average daily dose of 20 mg/kg BW tetraethylammonium salt of monensic acid for 14 days; Pb + Sal - Pb-exposed mice, treated with an average daily dose of 20 mg/kg BW tetraethylammonium salt of salinomycinic acid for two weeks. On the 29^th day of the experiment the samples (lungs and heart) were taken for atomic absorption analysis.ResultsThe results revealed that exposure of mice to Pb for 14 days significantly increased the concentration of the toxic metal in both organs and elevated the cardiac concentrations of Ca, Cu and Fe compared to untreated mice. Pb exposure diminished the lung concentrations of Ca and Zn compared to that of untreated controls. DMSA, monensin and salinomycin decreased the concentration of Pb in the lungs and heart. Among the tested chelating agents, only salinomycin restored the cardiac Fe concentration to normal control values.ConclusionThe results demonstrated the potential application of polyether ionophorous antibiotic salinomycin as antidote for treatment of Pb-induced toxicity in the lungs and heart. The possible complexation of the polyether ionophorous antibiotics with Ca(II) and Zn(II), which can diminish the endogenous concentrations of both ions in the lungs should be taken into account.     

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.     

Effect of dietary zinc level on serum carotenoid levels, body and shank pigmentation of chickens after experimental infection with coccidia

Two experiments were conducted to test the effects of a dietary zinc amino acid complex (Zn-AA) and an anticoccidial drug on Eimeria acervulina or Eimeria tenella infections. In each experiment, 288 day-old Three-Yellow-Chickens were used in a 2 x 3 factorial experimental design. Six groups were arranged randomly to receive three levels of Zn-AA (0, 40, or 80 mg/kg) alone or with salinomycin (60 mg/kg). Additionally an uninfected group was set as negative control. At the age of 21 days birds in Exp. 1 were inoculated with 3 x 10(4) sporulated E. acervulina oocysts, while birds in Exp. 2 were inoculated with 1.5 x 10(4) sporulated E. tenella oocysts. In Exp. 1, E. acervulina did not suppress growth performance significantly, but in groups without salinomycin it significantly reduced serum carotenoid levels on day 7 after inoculation and body and shank pigmentation on day 42. Salinomycin medication maintained serum carotenoids and visual colour of inoculated birds, but Zn-AA did not influence these parameters. In Exp. 2, growth performances of infected and uninfected chickens were similar. Infection decreased to only serum carotenoid levels on day 14 after infection, and colour scores on day 42 in the inoculated group without salinomycin and Zn-AA supplementation. The birds that received Zn-AA had significantly higher serum carotenoid levels and colour scores than those that did not. Although supplementation of Zn-AA cannot avoid coccidial damage of caecum, it prevents the reduction of serum carotenoids and pigmentation of Three-Yellow-Chicken infected with E. tenella, but not after infection with E. avervulina. The interactive effects between Zn-AA and salinomycin on growth performance and pigmentation were not significant.     

The action of salinomycin-Na and lasalocid-Na on chloroquine- and mepacrine-resistant line of Plasmodium berghei K 173-strain in Wistar rats

Salinomycin-Na and lasalocid-Na, two ionophorous antibiotics known for their anticoccidial activity, exhibit in vivo blood schizontocidal action on the Plasmodium berghei Keyberg 173 RC/M line that has a high level of resistance to chloroquine and mepacrine. Salinomycin was found to have a greater effect than lasalocid on asexual stages of this line. Trophozoites and schizonts were no longer found after a single dose of 20 mg/kg or five doses of 1.25 mg/kg of salinomycin whereas a single dose of 40 mg/kg or five doses of 20 mg/kg of lasalocid showed no marked effect on parasitaemia within 96 h of starting treatment in rats. Some toxicological data show that lasalocid, however, is better tolerated in domestic animals than salinomycin. Early morphological changes in asexual blood stages were membrane-coiling in the cytoplasm followed by vacuolization and disruption of the cell membrane or pellicle after treatment with both compounds. In particular mature schizonts were totally destroyed showing enormously large vacuoles. Toxicological data and blood schizontocidal activity indicate the narrow safety margin in P. berghei infected rats, and place salinomycin in the 'markedly toxic' group of antimalarial compounds.     

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.     

Identification and characterization of Eimeria tenella apical membrane antigen-1 (AMA1)

Apical membrane antigen-1 (AMA1) is a micronemal protein of apicomplexan parasites that appears to be essential during the invasion of host cells. In this study, a full-length cDNA of AMA1 was identified from Eimeria tenella (Et) using expressed sequence tag and the rapid amplification of cDNA ends technique. EtAMA1 had an open reading frame of 1608 bp encoding a protein of 535 amino acids. Quantitative real-time PCR analysis revealed that EtAMA1 was expressed at higher levels in sporozoites than in the other developmental stages (unsporulated oocysts, sporulated oocysts and second-generation merozoites). The ectodomain sequence was expressed as recombinant EtAMA1 (rEtAMA1) and rabbit polyclonal antibodies raised against the rEtAMA1 recognized a 58-kDa native parasite protein by Western Blotting and had a potent inhibitory effect on parasite invasion, decreasing it by approximately 70%. Immunofluorescence analysis and immunohistochemistry analysis showed EtAMA1 might play an important role in sporozoite invasion and development.     

The role of sialyl glycan recognition in host tissue tropism of the avian parasite Eimeria tenella

Eimeria spp. are a highly successful group of intracellular protozoan parasites that develop within intestinal epithelial cells of poultry, causing coccidiosis. As a result of resistance against anticoccidial drugs and the expense of manufacturing live vaccines, it is necessary to understand the relationship between Eimeria and its host more deeply, with a view to developing recombinant vaccines. Eimeria possesses a family of microneme lectins (MICs) that contain microneme adhesive repeat regions (MARR). We show that the major MARR protein from Eimeria tenella, EtMIC3, is deployed at the parasite-host interface during the early stages of invasion. EtMIC3 consists of seven tandem MAR1-type domains, which possess a high specificity for sialylated glycans as shown by cell-based assays and carbohydrate microarray analyses. The restricted tissue staining pattern observed for EtMIC3 in the chicken caecal epithelium indicates that EtMIC3 contributes to guiding the parasite to the site of invasion in the chicken gut. The microarray analyses also reveal a lack of recognition of glycan sequences terminating in the N-glycolyl form of sialic acid by EtMIC3. Thus the parasite is well adapted to the avian host which lacks N-glycolyl neuraminic acid. We provide new structural insight into the MAR1 family of domains and reveal the atomic resolution basis for the sialic acid-based carbohydrate recognition. Finally, a preliminary chicken immunization trial provides evidence that recombinant EtMIC3 protein and EtMIC3 DNA are effective vaccine candidates.     

In vivo action of the anticoccidial diclazuril (Clinacox) on the developmental stages of Eimeria tenella: a histological study

Diclazuril, a new benzeneacetonitrile anticoccidial, has potent activity against various stages of Eimeria tenella. A single treatment of experimentally infected chickens during the prepatent phase (up to day 5) results in a complete interruption of the life cycle and oocyst shedding. The first- and second-generation schizonts show extensive degenerative changes that finally result in a complete loss of the parasitic stage. The degeneration is characterized by loss of internal structure, the appearance of many intracytoplasmic vacuoles, and incomplete merogony. The merozoites themselves show similar degenerative changes, including the presence of numerous small vacuoles in the cytoplasm. Diclazuril is also effective against both the micro- and macrogametocytes that have a ballooned appearance and loose their internal structure completely. In the macrogametocytes, wall-forming bodies either do not develop or disappear rapidly. Development of typical caecal lesions is prevented when treatment with diclazuril is initiated before large numbers of second-generation schizonts appear, i.e., day 3. It is concluded that diclazuril is lethal against both the asexual and the sexual stages of E. tenella. At the proposed use level of 1 ppm in the feed, the life cycle is interrupted at a very early stage and lesion development and oocyst shedding are completely prevented.