Epidemiological investigation and drug resistance of Eimeria species in Korean chicken farms.

BACKGROUND: Coccidiosis is a poultry disease that occurs worldwide and is caused by Eimeria species. The infection is associated with reduced feed efficiency, body weight gain, and egg production. This study aimed to investigate the current status of coccidiosis and anticoccidial resistance to anticoccidial drugs used as part of control strategies for this disease in Korean chicken farms. RESULTS: An overall prevalence of 75% (291/388) was found. Positive farms contained several Eimeria species (mean = 4.2). Of the positive samples, E. acervulina (98.6%), E. maxima (84.8%), and E. tenella (82.8%) were the most prevalent species. Compared with cage-fed chickens, broilers and native chickens reared in free-range management were more at risk of acquiring an Eimeria infection. Sensitivities to six anticoccidial drugs (clopidol, diclazuril, maduramycin, monensin, salinomycin, and toltrazuril) were tested using nine field samples. Compared with untreated healthy control chickens, the body weight gains of infected chickens and treated/infected chickens were significantly reduced in all groups. Fecal oocyst shedding was significantly reduced in four clopidol-treated/infected groups, three diclazuril-treated/infected groups, two toltrazuril-treated/infected groups, one monensin-treated/infected group, and one salinomycin-treated/infected group, compared with the respective untreated/infected control groups. Intestinal lesion scores were also reduced in three clopidol-treated/infected groups, one monensin-treated/infected group, and one toltrazuril-treated/infected group. However, an overall assessment using the anticoccidial index, percent optimum anticoccidial activity, relative oocyst production, and reduced lesion score index found that all field samples had strong resistance to all tested anticoccidial drugs. CONCLUSION: The results of this large-scale epidemiological investigation and anticoccidial sensitivity testing showed a high prevalence of coccidiosis and the presence of severe drug resistant Eimeria species in the field. These findings will be useful for optimizing the control of coccidiosis in the poultry industry.

Microwave method synthesis of magnetic ionic liquid/gold nanoparticles as ultrasensitive SERS substrates for trace clopidol detection.

Clopidol is one of the most widely used anti-coccidiosis drugs. Its residues in poultry products and the environment pose a serious threat to human health. In this work, microwave-assisted synthesis of magnetic ionic liquid/gold nanoparticles (MIL-Au NPs) as the SERS substrates were first designed for sensitive and reliable determination of clopidol residue in egg samples. The experiment shows that MIL(1-methyl-3-hexyl imidazole ferric tetrachloride ([C6mim]FeCl4)) and microwave play a key role in the dispersion and morphology of Au NPs. Under the optimal conditions, the as-prepared MIL-Au NPs were applied to the SERS detection of clopidol in methanol and egg solution and its detection limits can be as low as to 0.5 µg/kg (equal to 0.5 ppb) in both solutions. The standard curves with regression coefficients of 0.9298 and 0.93496 were constructed in the linear range of 100-1000 ppb and 0.5-50 ppb for clopidol in egg solutions. Moreover, satisfactory recoveries (97.5-103.2%) were obtained for egg samples. The developed SERS method provides a way for quantitation of clopidol and can be applied for the convenient, reliable, and highly sensitive detection of antibiotic residues in food and environment, which has great potential in food safety and biological monitoring. Graphical abstract.

Development of an enzyme-linked immunosorbent assay for detection of clopidol residues in chicken tissues.

BACKGROUND: Clopidol is mainly used for the prevention and treatment of coccidiosis, which poses a serious potential hazard to public health, in veterinary medicine. The aim of this study was to prepare monoclonal antibodies (mAbs) against clopidol (CLOP) and develop an immunoassay for detecting CLOP residues in chicken tissues. After derivation, CLOP hapten was conjugated to carrier proteins to synthesize the artificial antigen, and immunized Balb/C mice were employed to screen mAbs. RESULTS: A sensitive hybridoma named C1G3 was screened out and two indirect competitive enzyme-linked immunosorbent assay (icELISA) standard curves were established. For the traditional two-step assay the linear range was from 0.06 to 98 ng mL(-1) , with half-maximal inhibitory concentration (IC50 ) and limit of detection (LOD) values of 2.76 ng mL(-1) and 0.03 ng mL(-1) respectively, while the rapid one-step icELISA had a working range from 0.08 to 102 ng mL(-1) , with IC50 and LOD values of 3.52 ng mL(-1) and 0.03 ng mL(-1) respectively. It was also indicated that a 10-fold dilution in chicken muscles gave an inhibition curve almost the same as that obtained in phosphate-buffered saline. When applied to spiking tests in chicken samples, the correlation coefficient (R(2) ) between concentrations added and measured was 0.9534. CONCLUSION: The results of this study suggest that the immunoassay described is a promising alternative for screening CLOP residues in biological matrices and is suitable for routine diagnostics.

Effect of butyrate, clopidol and their combination on the performance of broilers infected with Eimeria maxima.

1. The effect of butyric acid glycerides (BAGs) with and without clopidol (CLP) on Eimeria maxima on growth and associated biochemical variables was investigated in broiler chickens. 2. One-day-old chicks were divided into 6 equal groups (Gps) of 30 chicks each; each group was subdivided into 6 equal subgroups. Gp 1 was not infected and not treated. Chicks in Gp 2 were not infected and fed on a ration mixed with 4 g BAGs/kg for 6 successive weeks. Chicks of the other groups were directly inoculated intra-crop with 1 × 10(4) sporulated oocysts of E. maxima at 14 d of age. Gp 3 was infected and not treated. Chicks in the remaining three groups were given diets mixed with the tested drugs for 6 successive weeks. Gp 4 was fed on a diet mixed with CLP (125 g/kg). Gp 5 was given a diet mixed with BAGs (4 g/kg diet). Gp 6 was fed on a diet mixed with both BAGs (4 g/kg diet) and CLP (125 g/kg). 3. Birds in Gps 5 and 6 showed a reduction in the mean oocyst count, lesion scores and developmental stages in the lamina propria and improved growth and biochemical variables. BAG supplementation enhanced growth and production of healthy broilers. 4. It was concluded that BAGs were a useful supplement in broiler diets as an alternative to growth promoters and antimicrobial drugs.

Pathogenicity and drug resistance of the Eimeria tenella isolate from Yiwu, Zhejiang province, eastern China.

Chicken coccidiosis can cause severe enteritis with high mortality, which causes serious economic losses to the global breeding industry each year. The most virulent species is Eimeria tenella (E. tenella), but the infectivity of different E. tenella varies among geographic strains. At present, there are no reports related to the pathogenicity and drug resistance of E. tenella in Yiwu, Zhejiang province, China. A total of 600 fecal samples were collected from 10 farms in Zhejiang province, the overall oocyst prevalence was 54.2% (325/600). The prevalence was significantly higher (P < 0.01) in chickens under 40 d (97.5%) than that in chickens between 60 and 85-days-old (40.5%) and chickens over 90-days-old (24.5%). E. tenella stain was isolated from fecal samples of chickens in Yiwu and the pathogenicity of this isolate was determined, and then we recorded the survival rate, bloody stool score, lesion score, average weight gain. The results showed that all of the chickens infected with 5 × 105 sporulated oocysts of E. tenella died after the seventh day of infection, the bloody stool score and average lesion score of chickens from group 1 (5 × 105), group 2 (5 × 104), group 3 (5 × 103) and group 4 (5 × 102) decreased successively; the average weight gain (g) and relative weight gain (%) increased successively; the weight gain of the low-dose E. tenella infection groups (5 × 103 and 5 × 102) were higher than the other 2 groups (5 × 105 and 5 × 104) (P < 0.05). Finally, The E. tenella isolate was tested for sensitivity to 6 anticoccidial drugs (sulfachloropyrazine sodium, amproline, toltrazuril, clopidol, salinomycin, and nicarbazine) using 4 indexes including anticoccidial index(ACI), percent of optimum anticoccidial activity (POAA), reduction of lesion scores (RLS), and relative oocyst production (ROP). The results showed that this isolate has developed severe resistance to drugs of salinomycin and nicarbazine, moderate resistance to amproline and clopidol, slight resistance to toltrazuril, while the E. tenella isolate performed more sensitive to sulfachloropyrazine sodium.

Multispectral and molecular modeling investigations on the binding behaviors of two anticoccidials with serum albumins.

The interaction properties of monensin/clopidol with bovine/human serum albumin (BSA/HSA) were determined via multispectral together with molecular modeling techniques in the report. Fluorescence quenching spectra at different temperatures and fluorescence lifetime determination demonstrated that the fluorescence quenching belonged to a static quenching type. In the case of monensin-BSA, clopidol-BSA, monensin-HSA and clopidol-HSA, the binding constants Ka (291 K) were 5.42 × 104, 4.96 × 104, 3.22 × 104 and 2.99 × 104 M-1, respectively; the binding distances r0 were 1.88, 2.53, 2.19 and 2.02 nm, respectively. Monensin and clopidol bound strongly with BSA/HSA with binding free energies equal to -26.37/-25.11 and -26.11/-24.93 kJ mol-1, respectively. The spontaneous binding process was dominated by hydrogen bonds and van der Waals forces as reflected in thermodynamic parameters analyses. Synchronous, CD, FTIR and UV-vis spectra assays confirmed that serum albumins conformations were altered. Using competitive experiment, monensin/clopidol was observed to bind at site I of serum albumins, which were reconfirmed by the results of molecular modeling.Communicated by Ramaswamy H. Sarma.

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.

Determination of residual clopidol in chicken muscle by capillary gas chromatography/mass spectrometry.

Clopidol was extracted from chicken with acetonitrile, and the extract was evaporated to about 5 mL. The concentrated solution was centrifuged and applied to a glass column filled with basic alumina. The column was eluted with methanol and the methanol was evaporated to dryness. The residue was derivatized with Sylon BFT and analyzed by gas chromatography/mass spectrometry (GC/MS). Mass characteristics of trimethylsilyl derivative of clopidol were interpreted manually. The selected-ion monitoring mode was performed at m/z 191, 248, 263, and 265. A sensitive, reproducible GC/MS method was developed for monitoring residues of clopidol in chickens. All recoveries of the drug from chicken muscle fortified at 1, 10, and 100 ng/g were > 65%, and relative standard deviations were < 15%. The detection limit was about 0.5 ng/g, and the quantitation limit was the lower limit of the standard curve, which was about 1 ng/g.

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.

Model ecosystem evaluation of the environmental impacts of the veterinary drugs phenothiazine, sulfamethazine, clopidol, and diethylstilbestrol.

Four veterinary drugs of dissimilar chemical structures were evaluated for environmental stability and penchant for bioaccumulation. The techniques used were (1) a model aquatic ecosystem (3 days) and (2) a model feedlot ecosystem (33 days) in which the drugs were introduced via the excreta of chicks or mice. The model feedlot ecosystem was supported by metabolism cage studies to determine the amount and the form of the drug excreted by the chicks or mice. Considerable quantities of all the drugs were excreted intact or as environmentally short-lived conjugates. Diethylstilbestrol (DES) and Clopidol were the most persistent molecules, but only DES bioaccumulated to any appreciable degree. Phenothiazine was very biodegradable; sulfamethazine was relatively biodegradable and only accumulated in the organisms to very low levels. Data from the aquatic model ecosystem demonstrated a good correlation between the partition coefficients of the drugs and their accumulation in the fish.

Determination of clopidol residues in chicken tissues by high-performance liquid chromatography-mass spectrometry.

A high-performance liquid chromatographic-mass spectrometric (HPLC-MS) method has been developed for determination of clopidol residues in chicken tissues. Samples are extracted with acetonitrile. The extracts are cleaned up on an alumina column followed by an anion-exchange column. The clopidol is separated on a column (150 cmx4.6 mm) of Intertsil by using acetonitrile-water (20:80) as mobile phase. The clopidol was qualitatively identified by molecule mass and determined quantitatively by selected ion monitoring mode at 190 m/z. The recoveries with RSDs ranged from 91.6+/-10.1 to 97.3+/-5.7 at 0.010 to 10.0 mg/kg by spiking three matrices (chicken muscle, liver, and kidney). The limit of detection was 0.005 mg/kg, and the limit of quantification was 0.010 mg/kg.

Determination of nicarbazin and clopidol in poultry feeds by liquid chromatography.

A rapid and very effective analytical procedure for the simultaneous liquid chromatographic determination of two coccidiostats, clopidol (CLOP) and nicarbazin (NICA), in poultry feeds was developed and tested. The ground feed samples were extracted using aqueous dimethylformamide after moistening with water. Co-extracted feed constituents were removed with a solid-phase extraction alumina-basic column and the eluates were directly analyzed on an ODS column (250x4.6 mm, 5 microm) with acetonitrile-0.01 M acetate buffer (pH 4.6) as eluent. UV detection of CLOP and the 4,4'-dinitrocarbanilide portion of NICA was carried out at 265 and 345 nm, respectively. The mean recovery from NICA spiked samples was 95% with a RSD of 4% in a concentration range of 2-150 mg/kg while for CLOP it was 98% with a RSD of 5% in a concentration range of 5-150 mg/kg. The limits of detection of NICA and CLOP in feed, based on a detector signal-to-noise ratio of 3, were estimated to be 1 mg/kg and 2.5 mg/kg, respectively, and the lowest levels tested in feeds by this procedure were 2 mg/kg and 5 mg/kg, respectively.

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.

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.

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.

Development of immunity to coccidiosis in chickens administered anticoccidials in feed.

New combinations of techniques were employed for comparing the development of immunity of Eimeria tenella in chickens being medicated with 12 different anticoccidials. Broiler-type birds in batteries received a daily measured dose of E. tenella oocysts for 15 consecutive days while the drug was administered at the manufacturer's recommended level. Two or more tests of each drug gave the following ratings: strong suppression, monensin (121 ppm), salinomycin (80 ppm), lasalocid (75 ppm); moderate suppression, monensin (100 ppm), decoquinate (30 ppm), clopidol (125 ppm), and narasin (80 ppm); slight suppression, arprinocid (70 ppm), nicarbazin (125 ppm), and amprolium (125 ppm + ethopabate (4 ppm); no effect, robenidine (33 ppm), zoalene (125 ppm), and aklomide (250 ppm).

Efficacy of selected oral chemotherapeutants against Ichthyophthirius multifiliis (Ciliophora: Ophyroglenidae) infecting rainbow trout Oncorhynchus mykiss.

The chemotherapeutic efficacy of 6 in-feed compounds against Ichthyophthirius multifiliis Fouquet, 1876 was assessed using experimental infections of rainbow trout Oncorhynchus mykiss (Walbaum) fingerlings. Trial doses of 104 ppm amprolium hydrochloride or 65 ppm clopidol fed to fish for 10 d prior to infection significantly reduced the number of trophonts establishing in trout fingerlings by 62.0 and 35.2% respectively. In-feed treatments of infected trout with either 63 or 75 ppm amprolium hydrochloride, 92 ppm clopidol, or 38, 43 or 47 ppm salinomycin sodium for 10 d also significantly reduced the number of surviving trophonts by 77.6 and 32.2% for amprolium, 20.1% for clopidol and 80.2, 71.9 and 93.3% respectively for salinomycin sodium.

Eimeria stiedai in rabbits: the demonstration of responses to chemotherapy.

The activity of sulphaquinoxaline, robenidine, methyl benzoquate, clopidol and a mixture of methyl benzoquate and clopidol (Lerbek; Dow), was studied in rabbits infected with hepatic coccidiosis due to Eimeria stiedai. Growth inhibition, oocyst production and the activity in the serum of glutamate dehydrogenase and gamma glutamyltransferase were studied as indicators of parasite development. Only sulphaquinoxaline and Lerbek gave satisfactory control of this parasite. The latter formulation was more effective than either of its constituents used alone.