Relative virulences of a drug-resistant and a drug-sensitive strain of Eimeria acervulina, a coccidium of chickens.

The virulence of a field strain of the chicken coccidian parasite Eimeria acervulina (Boreham I), dually resistant to the chemically unrelated anticoccidial agents decoquinate and clopidol, was compared with that of a drug-sensitive laboratory strain (Ongar) of the same species. Following a single heavy infection (prevented from recycling), both strains exhibited pathogenic effects typical of their species, viz., pathognomonic lesions, adverse effects on body weight gain and feed conversion ratio (FCR), but no mortality. One week after infection, chicks infected with either strain had a statistically significantly worse weight gain than the uninfected control; the Boreham I strain produced more oocysts, and caused slightly more severe duodenal lesions and poorer FCRs than the Ongar strain (all those effects being non-significant). After 3 weeks, there were no significant differences between any cumulative effects of either strain, nor any differences from the uninfected control. However, from 2 to 3 weeks after infection, chicks infected with either strain had a greater feed consumption and growth rate than uninfected chicks. When chicks reared on solid floors were given lighter infections of either strain, which were allowed subsequently to recycle naturally, there were no consistent reductions in weight gains, but feed consumption was higher than that of uninfected chicks. Whatever, the mode of infection, there were no significant differences between the weights of infected and uninfected chicks after 3 weeks, but the FCR of infected chicks was usually poorer than that of uninfected chicks. The difference between the virulences of the Boreham I and Ongar strains was not greater than that between various drug-resistant strains or between various sensitive strains of several Eimeria species recorded in the literature. It is therefore concluded that there was no difference between the virulences of the two strains of E. acervulina that could be attributed to the drug-resistance of one of them.

Effects of decoquinate and clopidol on electron transport in mitochondria of Eimeria tenella (Apicomplexa: Coccidia).

Resistance of the chicken coccidium Eimeria tenella to the anticoccidial agents decoquinate and clopidol, and the synergistic activity of mixtures of these compounds have been confirmed in vivo. Inhibition of electron transport by decoquinate and clopidol has been studied in mitochondria isolated from unsporulated oocysts of the same lines of E. tenella as those used in the in vivo studies. Electron transport in mitochondria of a drug sensitive line was susceptible to inhibition by both decoquinate and clopidol, and mitochondria isolated from lines made resistant to one or the other of these compounds showed a corresponding resistance at the level of electron transport. Combinations of low concentrations of decoquinate and clopidol caused a greater inhibition of electron transport than expected from summation of their individual actions. Isobolograms showed that decoquinate and clopidol in fact potentiated each other's effect on electron transport. Induced resistance to either decoquinate or clopidol resulted in an increased sensitivity of electron transport to inhibition by the other drug. Cytochrome spectra of E. tenella mitochondria and a biphasic response of NADH-oxidase and terminal oxidase activity to inhibition by cyanide or azide suggest the presence of two functional terminal oxidases. There is a correlation between the resistance of electron transport to inhibition by decoquinate or clopidol and the susceptibility to inhibition by cyanide or azide. Mitochondrial electron transport that is more resistant to inhibition by decoquinate exhibits greater sensitivity to cyanide and azide; electron transport that is more resistant to inhibition by clopidol exhibits a decreased sensitivity to cyanide and azide. Resistance to decoquinate and clopidol is discussed in view of a possibly branched electron transport chain in mitochondria of E. tenella.

Mutagenicity screening of feed additives in the microbial system.

18 feed additives were tested for DNA-modifying effects by the repair test named "rec-assay" with Bacillus subtillis H17 (rec+) and M45 (rec-), and for mutagenicity with Escherichia coli WP2 hcr and 5 Salmonella typhimurium tester strains with the use of a top-agar overlay method. Carbadox, furazolidone, panazon and zoalene were positive in both assays. The former 3 were mutagenic for TA100, TA98 and WP2 hcr, while zoalene was mutagenic for all strains. These 4 compounds did not require a metabolic activation for their mutagenic activities. Nicarbazin was weakly mutagenic for TA1538 and TA98 with and without S9 mix. Amprolium and caprylohydroxamic acid also showed very weak mutagenicities only for TA100 with S9 mix and for WP2 hcr with and without S9 mix, resp. The mutagenic activities of carbadox, furazolidone and panazon for TA100 were reduced only by the addition of S9 mix, but not by S9 fraction or blood, whereas that of zoalene was decreased by any of the 3 factors.

Effect of clopidogrel on platelet aggregation and plasma concentration of fibrinogen in subjects with cerebral or coronary atherosclerotic disease.

Acetylsalicylic acid inhibits thromboxane A2 production and reduces the risk of vascular occlusive events by 20% to 25%. Ticlopidine inhibits ADP-dependent platelet aggregation and reduces the same risk by 30% to 35%, but produces some adverse effects. Clopidogrel is a ticlopidin-related antiplatelet drug, with the same mechanism of action; it reduces the expression of the glycoprotein IIb/IIIa, the fibrinogen receptor on the platelet surface. Clopidogrel has the same clinical efficacy of ticlopidin and has a decreased incidence of adverse effects. The effect of one daily dose of 75 mg of clopidogrel on platelet function in 90 subjects was evaluated; 41 with coronary artery disease and 49 with cerebral vascular disease. Before treatment and after 6 and 12 weeks, bleeding time and fibrinogen plasma concentration were also evaluated. There was a reduction in 5-microM ADP-induced platelet aggregation of 38%+/-27% at 6 weeks and 44%+/-29% at 12 weeks in patients with coronary artery disease; 35%+/-41%, 29%+/-59% in the cerebral vascular disease group; and 36%+/-36% and 35%+/-49% in the total group. Reduction of 20 microg/mL collagen-induced platelet aggregation was not significant in any group. Plasma fibrinogen levels did not vary during treatment. Bleeding time was significantly prolonged in all studied groups. There were no hemorrhagic complications; only digestive discomfort in less than 3% of patients. Clopidogrel efficiently reduces ADP-induced platelet aggregation and prolongs bleeding time and is a safe and efficacious antiplatelet drug.

Drug resistance in avian coccidia (a review).

Drug resistance is now recognised as a major cause of the failure of drugs to control coccidiosis in the fowl. In this article, biological, biochemical and genetic aspects of resistance in Eimeria are reviewed and some of the problems that may limit progress in understanding the nature of resistance in coccidia are discussed.

Effects of clopidol on sporulation and infectivity of Eimeria tenella oocysts.

Feed additive anticoccidials currently used in Japan were examined for possible effects on oocyst sporulation of Eimeria tenella. Monensin, salinomycin, lasalocid, amprolium plus ethpabate, amporolium plus ethopabate plus sulfaquinoxaline, clopidol, or nicarbazin were given to chickens continuously via the feed at the recommended use level or one-half of that level. Oocysts discharged in feces 7-8 days post inoculation (PI) were collected and aerated for sporulation. Low sporulation rate was noted, when clopidol at 62.5 mg kg-1 was given from 4 to 7 days PI. These oocysts were as infective as oocysts from controls, based on weight gain, feed efficiency, gross lesion score of cecae, and oocyst count 7 days PI. The results of the study indicated that the second schizogony and gametogony are vulnerable to clopidol, as evidenced by oocyst sporulation, but infectivity of these sporulated oocysts was not affected.

Comparative efficacy of anticoccidials under the conditions of commercial broiler production and in battery trials.

Coccidia were isolated from a commercial broiler farm with a history of suspected drug resistance. The sensitivity profiles of the Eimeria spp. isolates against the anticoccidial drugs nicarbazin (NIC), narasin (NAR), halofuginone (HAL), salinomycin (SAL), meticlorpindol plus methylbenzoquate (MET), and monensin (MON) at the recommended dose levels were followed in three battery trials (B1, B2, B3) corresponding to a field study over three periods of commercial broiler keeping (F1, F2, F3). Shuttle programs were performed in F1 (NIC/MON) and in F2 (MET/MON) while only SAL was used in F3. Eimeria acervulina and E. tenella were isolated from indicator birds in F1 while only E. acervulina could be found during F2 and F3. In trial B1 the isolate from F1 was identified as resistant against HAL and partly resistant against NIC and MON, the two drugs that were used in F1. Following the replacement of NIC in the starter feed by MET the respective isolate from F2 showed no resistance against ionophores (trial B2) while partial resistance against HAL was still present. Since SAL was the most efficient drug in B1 and B2 only this drug was applied in F3. Apart from a resistance against HAL no resistance against any of the other tested anticoccidials was found in the isolate from F3. SAL controlled coccidiosis efficiently in the field and best productivity was recorded in F3. This study shows that battery trials have a good predictive value in respect to the efficacy of anticoccidials under the conditions of commercial broiler production.

Immunity and effect of clopidol/methyl benzoquate and robenidine before and after weaning on rabbit coccidiosis in the field.

For 15 months the anticoccidial effect of 200 ppm clopidol/methyl benzoquate and of 50 ppm robenidine, and the development of immunity against five different species of Eimeria were followed in a closed rabbit population. In unmedicated rabbits, oocyst output decreased progressively with increasing age to a very low level in animals older than four months, but none of the species present disappeared completely in adult animals. No clinical symptoms nor mortality from coccidiosis was noted in reproduction stock. In field conditions E magna and E perforans seemed to induce the weakest resistance, whereas a more marked resistance has been found for E intestinalis and E irresidua. E media appeared to have an intermediate position. Robenidine reduced oocyst output of E magna, E intestinalis, E irresidua, E media and E perforans significantly, whereas clopidol/methyl benzoquate reduced oocyst output of the latter four species only and was least active against E magna. Both drugs also reduced coccidiosis-induced mortality significantly. Medication only before weaning had no distinct influence on coccidial infection, or on mortality by coccidiosis after weaning; nor did those parameters differ significantly between continuously medicated rabbits and rabbits medicated after weaning only. As reproductive stock is protected by immunity, this makes the necessity of medicating does and bucks with anticoccidials questionable in intensive or semi-intensive reproduction systems.

Sensitivity of field isolates of Eimeria tenella to anticoccidial drugs in the chicken.

Thirty isolates of Eimeria tenella obtained from broiler and breeder farms were examined for their sensitivity to anticoccidial drugs. All were sensitive to robenidine, 28 were sensitive to methyl benzoquate, 25 to clopidol and 21 to nicarbazin. Most isolates were resistant or partly resistant to amprolium and dinitolmide.

Lasalocid: resistance and cross-resistance studies in Eimeria tenella-infected chicks.

Eimeria tenella was passaged in the presence of suboptimal and optimal concentrations of lasalocid (X-537A, sodium salt) in feed. Lasalocid was equally active at concentrations of 0.006 and 0.0075% against the 10th and 15th passage of E. tenella lasalocid exposed strains. Resistance to lasalocid could not be induced. Lasalocid administered in the feed at 0.0075% was tested in controlled battery experiments against E. tenella strains resistant to known anticoccidials in chicks. These studies demonstrated that lasalocis, at the optimal feed concentration of 0.0075% was highly effective against coccidiosis induced by strains of E. tenella resistant to sulfaquinoxaline, nicarbazine, zoalene, emprolium, clopidol and 4-hydroxyquinoline. Lasalocid medicated chicks were heavier, converted feed more efficiently, showed less pathologic lesions, and had lower mortality (P less than or equal to .05) than the infected unmedicated controls as well as sulfaquinoxaline, nicarbazine, zoalene, amprolium-ethopabate, clopidol, buquinolate, decoquinate and nequinate medicated groups. Cross-resistance to lasalocid was not demonstrated.

Antimalarial quinolones: synthesis, potency, and mechanistic studies.

In the present article we examine the antiplasmodial activities of novel quinolone derivatives bearing extended alkyl or alkoxy side chains terminated by a trifluoromethyl group. In the series under investigation, the IC50 values ranged from 1.2 to approximately 30 nM against chloroquine-sensitive and multidrug-resistant Plasmodium falciparum strains. Modest to significant cross-resistance was noted in evaluation of these haloalkyl- and haloalkoxyquinolones for activity against the atovaquone-resistant clinical isolate Tm90-C2B, indicating that a primary target for some of these compounds is the parasite cytochrome bc1 complex. Additional evidence to support this biochemical mechanism includes the use of oxygen biosensor plate technology to show that the quinolone derivatives block oxygen consumption by parasitized red blood cells in a fashion similar to atovaquone in side-by-side experiments. Atovaquone is extremely potent and is the only drug in clinical use that targets the Plasmodium bc1 complex, but rapid emergence of resistance to it in both mono- and combination therapy is evident and therefore additional drugs are needed to target the cytochrome bc1 complex which are active against atovaquone-resistant parasites. Our study of a number of halogenated alkyl and alkoxy 4(1H)-quinolones highlights the potential for development of "endochin-like quinolones" (ELQ), bearing an extended trifluoroalkyl moiety at the 3-position, that exhibit selective antiplasmodial effects in the low nanomolar range and inhibitory activity against chloroquine and atovaquone-resistant parasites. Further studies of halogenated alkyl- and alkoxy-quinolones may lead to the development of safe and effective therapeutics for use in treatment or prevention of malaria and other parasitic diseases.

The activity of methyl benzoquate and clopidol against Eimeria maxima: synergy and drug resistance.

Synergy between clopidol and methyl benzoquate against Eimeria maxima was shown to be supra-additive. Collateral sensitivity to these drugs could not be demonstrated in resistant lines of this parasite. Resistance to methyl benzoquate and clopidol was not transferred when lines of E. maxima, resistant to the respective drugs, were propagated together. The failure to demonstrate this phenomenon was judged not to be due to synergy between the drugs. Attempts to induce simultaneous was readily acquired by a line of E. maxima resistant to clopidol. Induced resistance to clopidol in a methyl benzoquate-resistant line required numerous passages.

Transferred drug-resistance in Eimeria maxima.

A series of experiments is described in which two drug-resistant strains of Eimeria maxima were passaged together in untreated chicks. The resultant oocysts were then inoculated into chicks treated with both drugs. When strains resistant to methyl benzoquate and sulphaquinoxaline or clopidol and sulphaquinoxaline were used the resultant infections were not controlled by the double treatment, indicating the acquisition of resistance factors by one strain from the other. When strains resistant to clopidol and methyl benzoquate were used the phenomenon was not observed.

The development of drug-resistant strains of Eimeria maxima in the laboratory.

The development of strains of Eimeria maxima resistant to buquinolate, methyl benzoquate, clopidol, sulphaquinoxaline and robenidine is described. It was not possible to standardize a schedule of inoculations and drug administration, which would enable the development of resistance to the different drugs to be compared directly. Resistance developed most readily to the quinolones. One robenidine-resistant strain proved to be drug-dependent. Dinitolmide showed unusual effects upon sporogony and three attempts to develop resistance against this activity failed. Chicks previously immunized with the parent strain were completely protected against infection with the drug-resistant strains.

Further studies on the use of chicken embryo infections for the study of drug resistance in Eimeria tenella.

Infections in the chicken embryo have been used to study the development of drug resistance in an embryo adapted strain of E. tenella. Resistance was developed to decoquinate, clopidol and robenidine by serially passaging this strain, but evidence for the development of resistance to amprolium was inconclusive. Resistance to decoquinate developed more readily than to the other drugs. Attempts to increase resistance to clopidol, robenidine and amprolium by increasing the sporozoite inoculum and by the use of a mutagenic agent were unsuccesful. No cross-resistance was found between the 4 drugs. Drug resistant lines of the Houghton strain (H) of E. tenella, made resistant to the 4 anticoccidial drugs by passage in chickens, were found to be resistant when evaluated using chicken embryo infections. Lines made resistant to decoquinate were not controlled by any concentration of this drug, suggesting that resistance, once developed, was absolute and not dependent on drug concentration. Lines made resistant to robenidine, clopidol and amprolium, however, were controlled by higher drug concentrations suggesting that in this case resistance was dependent on drug concentration.

Development by genetic recombination of a line of Eimeria tenella resistant to robenidine, decoquinate and amprolium.

A line of Eimeria tenella (H) resistant to robenidine, decoquinate and amprolium has been produced by genetic recombination. It was not possible to obtain a cross between this line and lines resistant to clopidol or arprinocid and halofuginone. Recombination between lines resistant to arprinocid and halofuginone resulted in a loss of pathogenicity.

Eimeria tenella: experimental studies on the development of resistance to amprolium, clopidol and methyl benzoquate.

The development of resistance by the Houghton strain of Eimeria tenella to the anticoccidial drugs amprolium, clopidol and methyl benzoquate has been studied. Resistance to amprolium and clopidol developed more readily in experiments where a large number of coccidia were exposed to the drug, either by increasing the number of oocysts in the inoculum or by increasing the number of birds in the group. When 45 birds were given 2.0 X 10(6) oocysts, resistance to amprolium and clopidol appeared after 6 and 7 passages respectively. In previous experiments, under similar conditions, resistance to robenidine developed after 6 passages, suggesting little difference between these three drugs. Resistance to amprolium and clopidol arose gradually as the concentration of drug was increased, but resistance to methyl benzoquate appeared in a single step from sensitivity to high-level resistance. Both amprolium and clopidol-resistant lines showed an 8-fold reduction in drug sensitivity. Attempts to measure the degree of resistance by calculation of the ED50 were unsuccessful.

Influence of some coccidiostatics on blood carotenoids and fatty acids in coccidiosis affected chicken.

Blood and liver carotenoids and A-vitamin and blood fatty acid modifications were observed in Eimeria tenella infested chicken. 125 chicken were alloted in 5 groups: 3 infested groups were given Amprol, Suldrazin and Clopindol respectively, in their diet. A control group was infested, a second one non-infested. Biochemical examinations were performed 10 days following treatment when chicken were slaughtered and samples collected. In control groups and in Amprol and Clopindol treated groups were seen significant decreases of liver and blood carotenoids; blood and liver A-vitamin levels were higher in Amprol treated chicken; in Suldrazin treated group no significant modifications could be shown as compared to the non-infested controls. Blood fatty acid levels were higher (43.5-47.8%) in the four infested groups and lower in the non-infested controls (39.4%). Non-saturated fatty acid levels varied within 51 and 56.4% in infested groups.