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.     

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.     

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.     

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.     

Effect of the anticoccidial arprinocid on production, sporulation, and infectivity of Eimeria oocysts

Medication of broilers with arprinocid [MK-302, 9-(2-chloro-6-fluorbenzyl adenine)] had 3 distinct effects on oocysts; (1) the number of oocysts produced was decreased, (2) fewer of the oocysts sporulated, and (3) those oocysts which did sporulate were less infective than those from unmedicated birds. The drug level necessary to prevent passage of oocysts depended on the species and strain of coccidia. To essentially eliminate oocyst production (less than 5% of controls) required medication with the following levels of arprinocid: 70 ppm with Eimeria maxima; 60 ppm with E. mivati, E. E. necatrix, and E. brunetti; and 50 ppm with E. tenella. With E. acervulina, oocysts were completely eliminated by 60 ppm of arprinocid with one field strain but were still numerous at 70 ppm with a second field strain. Oocysts recovered from birds on medication often failed to sporulate. No sporulation was seen at drug levels of 30 ppm or above with E. maxima and E. mivati. The level of arpinocid required to prevent sporulation with other species depended on the strain being studied, but varied from 30 ppm to 70 ppm. The oocysts of E. acervulina, E. mivati, E. tenella, and E. brunetti recovered from medicated birds that subsequently sporulated, were less infective when inoculated into susceptible birds, than oocysts from unmedicated birds. Oocysts from low medication level with E. necatrix (30 ppm) and E. maxima (10 ppm), once sporulated, were as infective as oocysts from unmedicated control birds, even though the numbers produced were less. No differences were detected in the time oocysts were produced between medicated and unmedicated birds infected with E. acervulina, E. maxima, E. brunetti, and E. tenella.     

The action of polyether ionophorous antibiotics (monensin, salinomycin, lasalocid) on developmental stages of Eimeria tenella (Coccidia, Sporozoa) in vivo and in vitro: study by light and electron microscopy

The effect of three polyether antibiotics (monensin, salinomycin, lasalocid) on developmental stages of Eimeria tenella (Coccidia, Sporozoa) was studied in vivo and in vitro by means of light and electron microscopy. It was found that these three drugs act against free merozoites, which are destroyed by bursting of the cell border (i.e. pellicle), endoplasmic reticulum and internal organelles even after very short exposure times (20 min) in media containing 1 ppm, 10 ppm or 100 ppm of these drugs. Sporozoites, however, survived these drug concentrations during an exposure time of 30 min (this would be sufficient to penetrate host cells and start development). Intracellular stages, which were situated in a parasitophorous vacuole within an intact host cell, were not attacked, apparently because these drugs are almost incapable of penetrating host cells. On the other hand, parasites (such as differentiated schizonts, gamonts) located within degenerating host cells showed slight disintegration, which did not necessarily led to their death. From these results it becomes clear why these polyether antibiotics have to be fed daily. Doses of 70 ppm salinomycin, 125 ppm monensin and 125 ppm lasalocid were found to bring about an equivalent protective effect against an infection with 40,000 Eimeria tenella oocysts.     

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.     

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.     

Electrophoretic and immunologic characterization of proteins of merozoites of Eimeria acervulina, E. maxima, E. necatrix, and E. tenella.

Merozoites of Eimeria acervulina, Eimeria maxima, Eimeria necatrix, and Eimeria tenella were compared by gel electrophoresis, western-blotting with chicken antiserum, indirect fluorescent antibody reactions, and antiserum neutralization. Merozoites from the 4 species had dissimilar patterns of proteins and antigens in soluble and membrane fractions. Coomassie blue staining of SDS-PAGE gels revealed 16-22 protein bands depending on the species of merozoite but only 3 bands per species in the membrane fractions. Homologous and heterologous antisera recognized 5-12 soluble fraction bands and 3-7 membrane fraction bands on immunoperoxidase-stained western blots, depending on the species. When antisera from infected chickens were used in an indirect fluorescent antibody reaction, the merozoites of E. tenella and E. necatrix had a strong reaction with homologous and heterologous antisera. Merozoites of E. acervulina and E. maxima reacted with homologous antisera but had a weak or no reaction with heterologous antisera. Chicken antiserum against E. tenella had no effect on the viability of E. tenella merozoites when they were inoculated into chicken embryos.     

Molecular characterization and phylogenetic analysis of Eimeria from turkeys and gamebirds: implications for evolutionary relationships in Galliform birds

In order to determine the evolutionary relationships among Eimeria species that parasitize birds of the Galliformes, the 18s rDNA gene and a portion of the cytochrome oxidase subunit 1 (cox-1) were amplified from Eimeria species isolated from turkeys, chukars, and pheasants. The phylogenetic analysis of these sequences suggests that species infecting chickens are polyphyletic and, therefore, do not all share a direct common ancestor. Both the 18s rDNA and the cox-1 sequences indicate that Eimeria tenella and Eimeria necatrix are more closely related to Eimeria of turkeys and pheasants than to other species that infect the chicken. It is, therefore, likely that the chicken Eimeria spp. represent 2 separate ancestral colonizations of the gut, one of which comprises E. tenella and E. necatrix that infect the ceca, while the other includes Eimeria acervulina, Eimeria brunetti, Eimeria maxima, and Eimeria mitis, which infect the upper regions of the intestine.     

Efficient and sensitive detection of residues of nine coccidiostats in egg and muscle by liquid chromatography-electrospray tandem mass spectrometry

We present a method based on electrospray liquid chromatography tandem mass spectrometry (LC-MS/MS) for determining in muscle and eggs the following nine coccidiostats: halofuginone, diclazuril, dinitrocarbanilide (the main metabolite of nicarbazin), robenidine, monensin, lasalocid, narasin, salinomycin, and maduramicin. Dinitrocarbanilide-d8, nigericin, and diclazuril-bis were used as internal standards. The method uses extraction in acetonitrile followed by a clean-up on an SiOH solid-phase extraction column. High-performance liquid chromatography (HPLC) separation was performed on a Purospher C(18) column (125 mm x 3 mm i.d.) protected by a guard column, the mobile phase being a water-acetonitrile gradient (each gradient component containing 0.1% formic acid) at a flow rate of 1 ml min(-1). For unequivocal identification of each analyte, two ions were detected and chosen for multiple reaction monitoring (MRM). Validation was carried out on spiked muscle and egg samples. The method described meets all the criteria of Decision 2002/657/EC and is easy to use in routine analysis. Validation results are presented with the measured CCalpha and CCbeta values. This whole method allows extraction and analysis of up to 24 samples per day.     

Eimeria tenella and E. necatrix: a third generation of schizogony is an obligatory part of the developmental cycle

The latter part of endogenous development of Eimeria tenella and E. necatrix was examined in sections of cecal tissue taken from chickens infected either by giving oocysts orally or by injecting merozoites into the cecum. The findings, with 3 strains of E. tenella and 1 of E. necatrix, indicate that the majority, if not all, of the parasite populations undergo a third generation of schizogony and then embark upon gametogony.     

Eimeria spp. of the domestic fowl: resolution of chromosomes by field inversion gel electrophoresis

The molecular karyotypes of five species of chicken coccidia, viz., Eimeria acervulina, E. brunetti, E. maxima, E. necatrix, and E. tenella, were determined using field inversion gel electrophoresis (FIGE). Each species has a distinctive set of resolvable chromosomes which range from about 1 to greater than 5.7 megabases. We were able to resolve at least 8 chromosomes for E. acervulina, 5 for E. brunetti, 10 for E. maxima, 6 for E. necatrix, and 9 for E. tenella. If the value of 67 megabases for the genomic DNA of E. tenella is accurate, then under the conditions used here only about 60% of its chromosomal complement has been resolved.     

Efficacy and cross-resistance studies on N, N'-bis (3,4 ditrifluoromethylphenyl) methylmalonamide, a novel anticoccidial agent.

N,N'-bis (3,4 ditrifluoromethylphenyl) methylmalonamide (Sch 18545) completely controlled a mild Eimeria necatrix infection at 50, 40 or 30 p.p.m. in the diet, and controlled E. tenella infections at 50 and 40 p.p.m. Slight oocyst passage was observed at each E. tenella treatment level with a marked increase at the 30 p.p.m. treatment level. Fifty p.p.m. were necessary to control E. acervulina infections; levels of 40 p.p.m. reduced E. acervulina oocyst production while 30 p.p.m. were ineffective. Evaluations of Sch 18545 using a mixed infection (Coccivac D) further suggested that activity with this compound was weakest against E. acervulina. Weight gains decreased with increasing concentration of drug in the diet of treated, infected birds and thus the compound showed an insufficient safety margin to be of practical value. Such 18545 administered at 35 p.p.m. in the diet was effective against amprolium, zoalene, aklomide or nicarbazin-resistant strains of E. tenella.     

Effect of coccidiosis on blood coagulation in broilers

Severe infection with Eimeria acervulina, Eimeria maxima, Eimeria necatrix, and Eimeria tenella increased the prothrombin times in broilers compared with the times in uninfected birds. Recalcification time was not affected. The increase in prothrombin time was related to the severity of infection (as measured by lesion score), and was significant (P less than or equal to 0.05) only in the most severely infected birds. The increase was of short duration, lasting only 1 or 2 days, and first appeared on day 5 or 6 postinoculation. Restricting the feed intake of uninoculated birds to the amount of feed consumed by infected birds showed that the reduction in feed intake with coccidiosis was not responsible for the increase in prothrombin time.     

Eimeria tenella: genomic organization and expression of an 89kDa cyclophilin

Though parasite cyclophilins are promising new drug targets, Eimeria tenella cyclophilins have not been characterized yet. Here, we describe an 89kDa cyclophilin, designated EtCYP89. It is expressed throughout the developmental cycle of E. tenella, both in the intracellular stages in chicken and in extracellular sporulated oocysts and sporozoites. The EtCYP89 protein contains two Ser-rich domains in its NH2-terminus separated by a His-rich stretch. WD40 repeats are localized in the central part of the protein followed by a cyclophilin domain at the COOH-terminus. Both protein and genomic organization of EtCyp89 are conserved in comparison with its ortholog TgCyp81.6 in Toxoplasma gondii, except for the absence of a Ser- and His-rich NH2-terminus in TgCYP81.6. In particular, those 13 residues are conserved which are responsible for binding the anti-coccidial drug cyclosporine A.     

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.     

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.     

Characterization of the complete mitochondrial genomes of five Eimeria species from domestic chickens

Chicken coccidiosis caused by members of the genus Eimeria causes significant economic losses worldwide. In the present study we sequenced the complete mitochondrial DNA (mtDNA) sequences of six Eimeria species and analyzed features of their gene contents and genome organizations. The complete mt genomes of E. acervulina, E. brunetti, E. maxima, E. necatrix, E. tenella and E. praecox were 6179bp, 6148bp, 6169bp, 6214bp, 6213bp and 6174bp in size, respectively. All of the mt genomes consist of 3 genes for proteins (cox1, cox3, and cytb), 12 gene fragments for the large subunit (LSU) rRNA, and 7 gene fragments for the small subunit (SSU) rRNA, but no transfer RNA genes. The organization of the mt genomes is similar to that of Plasmodium, but distinct from Babesia and Theileria. The putative direction of translation for 3 genes (cox1, cox3, and cytb) was the same in all six Eimeria species. The contents of A+T of the mt genomes were 65.35% for E. acervulina, 65.43% for E. brunetti, 64.53% for E. maxima, 65.04% for E. necatrix, 64.98% for E. tenella and 65.59% for E. praecox. The AT bias has a significant effect on both the codon usage pattern and amino acid composition of proteins. Phylogenetic analyses using concatenated nucleotide sequences of the 2 protein-coding genes (cytb and cox1), with three different computational algorithms (Bayesian analysis, maximum parsimony and maximum likelihood), all revealed distinct groups with high statistical support, indicating that the six Eimeria spp. represent six distinct but closely-related species. These data provide novel mtDNA markers for studying the molecular epidemiology and population genetics of the six Eimeria spp., and should have implications for the molecular diagnosis, prevention and control of coccidiosis in domestic chickens.     

Effect of Antibiotic Growth Promoters and Anticoccidials on Growth of Clostridium perfringens in the Caeca and on Performance of Broiler Chickens

The effects of the growth promoters avoparcin and avilamycin and the ionophore anticoccidials maduramicin, narasin and monensin on the growth of Clostridium perfringens (Cp) in the ceaca and on performance of broiler chickens were tested in 2 experiments. The supplements were fed as single feed additives or in some combinations. No clinical signs or lesions caused by coccidia were observed in any of the studies. All supplements had an antibacterial effect on Cp and improved growth rate significantly. Carcass yield of birds fed growth promoters avilamycin or avoparcin was significantly higher compared with birds fed anticoccidials. These data indicate that, what concerns bird performance, during good hygienic conditions supplementation with antibiotic growth promoters may not be necessary when the diet is supplemented with an anticoccidial with antibacterial effects.