In vitro anti-Toxoplasma gondii effects of a coccidiostat dinitolmide.

Coccidiosis, caused by Eimeria species, results in huge economic losses to the animal industry. Dinitolmide, a veterinary-approved coccidiostat, has a wide anticoccidial spectrum with no effect on host immunity. However, the mechanism of its anticoccidial effects remains unclear. Here, we used an in vitro culture system of T. gondii to explore the anti-Toxoplasma effect of dinitolmide and its underlying mechanism against coccidia. We show that dinitolmide has potent in vitro anti-Toxoplasma activity with the half-maximal effective concentration (EC50) of 3.625 µg/ml. Dinitolmide treatment significantly inhibited the viability, invasion and proliferation of T. gondii tachyzoites. The recovery experiment showed that dinitolmide can completely kill T. gondii tachyzoites after 24 h of treatment. Morphologically abnormal parasites were observed after dinitolmide exposure, including asynchronous development of daughter cells and deficiency of parasite inner and outer membrane. Further electron microscopy results showed that the drug could damage the membrane structure of T. gondii. By comparative transcriptomic analysis, we found that genes related to cell apoptosis and nitric-oxide synthase were up-regulated after dinitolmide treatment, which might be responsible for parasite cell death. Meanwhile, many Sag-related sequence (srs) genes were down-regulated after treatment, which could be closely associated with the reduction of parasite invasion and proliferation capacity. Our study indicates that the coccidiostat dinitolmide has a potent inhibitory effect on T. gondii in vitro and provides insight into the mode of action of the drug.

Electrochemical determination of dinitolmide in poultry product samples using a highly sensitive Mn2O3/MCNTs-NPs carbon paste electrode aided by greenness assessment tools.

In this paper, chemically modified carbon paste Mn2O3/MCNTs-NPs electrode for estimation of dinitolmide (DOM) utilizing square wave voltammetry method (SWV) was developed. The study investigated the electrochemical behavior of DOM, and the morphology of the modified electrode was evaluated by Scanning Electron Microscope (SEM) and Transmission Electron Microscope (TEM). The voltammetric behavior of DOM at modified electrode was recorded at a scan rate of 100 mVs-1 against Ag/AgCl reference electrode in phosphate buffer pH 4.0 within linearity range 2-12 µM, LOQ, and LOD of 1.8 and 0.594 µM, respectively, with average % recovery of (100.89 ± 0.795). GAPI and Analytical Eco-Scale tools were applied for greenness assessment. Specificity and interference study was valid for the proposed method; allowing DOM to be determined in its acidic degradation and its major interference drug. The proposed method was successfully employed to quantify DOM in bulk powder, egg, and frozen cuts-up chicken muscle samples.

Development of a gold nanoparticle-based lateral-flow strip for the detection of dinitolmide in chicken tissue.

Dinitolmide is a nitro amide coccidiostat used in poultry feed, and is a potential threat to the environment and human health. In this study, a monoclonal antibody (mAb) against dinitolmide was prepared and an immunochromatographic assay (ICA) was developed to detect residual dinitolmide in chicken tissue. The results show that the mAb exhibited high sensitivity, with a limit of detection as low as 9.01 ng mL-1. A cross reactivity test revealed that the mAb also had good specificity for dinitolmide. This ICA method showed a visible limit of detection of 2.5 µg kg-1, and a cut-off value of 25 µg kg-1 for testing dinitolmide in chicken sample extract by using the naked eye. Importantly, these observations using our ICA, were comparable to other methods of detection such as liquid chromatography, tandem mass spectrometry and indirect competitive enzyme-linked immunosorbent assays (IC-ELISA). These data suggest that our ICA method is a reliable, portable, and high-throughput method for the detection of dinitolmide residues in chicken tissue.

Comparative evaluation of herbal coccidiostat with chemotherapeutic coccidiostats on performance of broilers to control coccidiosis.

The objective of the present study was to investigate the comparative efficacy of recommended dose of selected anticoccidial drugs Salinomycin, Dinitolmide, while Cocciban at three dose levels on the performance of broilers. For this purpose, 420-day-old commercial male broiler chicks were randomly divided into 7 treatment groups with 10 replications of 6 birds each and reared in battery brooders up to 42 days of age. Groups were designated as uninfected unmedicated (T1), infected unmedicated (T2), Cocciban 500 g/ton and infected (T3), Cocciban 750 g/ton and infected (T4), Cocciban 1000 g/ton and infected (T5), Salinomycin 500 g/ton and infected (T6), and Dinitolmide and infected (T7). Groups T2, T3, T4, T5, T6, and T7 were experimentally infected at 21 days old by 50,000 oocysts of Eimeria species. The broilers were fed with starter (0-21 days) and finisher diets (22-42 days). The herbal product Cocciban 1000 g/ton alone had significantly (P < 0.05) higher body weight gain and feed efficiency than all other infected groups during the overall experimental period (0-42 days), but significantly lower than healthy control. All the groups did not show significant (P > 0.05) effect on mean feed intake, percent carcass yields and percent weights of liver, heart and gizzard. Similarly there was no significant (P < 0.05) influence of treatment groups on the organoleptic characteristics of meat. Treatment groups did not have any significant (P < 0.05) influence on humeral immune response to ND vaccine and cell-mediated immune response to PHA-P. Among all the infected groups, Cocciban 1000 g/ton group (78.33%) recorded more mean percent livability than all other infected groups.

Effect of Cocciban herbal coccidiostats on hematobiochemical, fecal parameters and cecal histopathology of broiler chicken.

The objective of the present study was to investigate the comparative efficacy of recommended dose of selected anticoccidial drugs Salinomycin and Dinitolmide, while Cocciban at three dose levels on the hematobiochemical, fecal parameters and histopathology of broilers. For this purpose, 420-day-old commercial male broiler chicks were randomly divided into 7 treatment groups with 10 replications of 6 birds each and reared in battery brooders up to 42 days of age. Groups were designated as uninfected unmedicated (T1), infected unmedicated (T2), Cocciban 500 g/ton and infected (T3), Cocciban 750 g/ton and infected (T4), Cocciban 1000 g/ton and infected (T5), Salinomycin 500 g/ton and infected (T6), and Dinitolmide and infected (T7). Groups T2, T3, T4, T5, T6, and T7 were experimentally infected at 21-day-old by 50,000 oocysts of Eimeria species. The mean fecal, lesion scores and oocyst per gram of feces were significantly (p < 0.05) highest in infected unmedicated group, while lowest in the herbal Cocciban 1000 g/ton group than all other infected medicated groups. The hematological studies revealed a reduction in TEC, Hb, and PCV from 0 to 5th day of P.I. in all infected groups except healthy control group. The birds of all the infected groups improved in the values of TEC, Hb, PCV, blood glucose, and total serum protein on 7th day of P.I., but, the improvement was significantly (p < 0.05) better in herbal Cocciban 1000 g/ton treated birds than all other infected groups. Whereas, the TWBC counts were raised from 0 to 7th day of P.I. in all the infected groups compared to healthy control and no significant (p < 0.05) difference was observed in between the infected groups. The histopathological changes consisting of desquamation of epithelial cells, cellular infiltration, hemorrhages, edema, fibrous tissue proliferation, and developing stages of E. tenella at various depths of cecal wall were higher in all the infected groups when compared to Cocciban 1000 g/ton group.

Study on the interaction of amprolium HCl and dinitolmide in animal-derived food products with BSA by multiple spectroscopies and molecular modeling techniques.

Communicated by Ramaswamy H. Sarma.

Development of an ASE-GC-MS/MS method for detecting dinitolmide and its metabolite 3-ANOT in eggs.

An accelerated solvent extraction coupled with gas chromatography-tandem mass spectrometry (ASE-GC-MS/MS) method for detecting dinitolmide residue and its metabolite (3-amino-2-methyl-5-nitrobenzamide, 3-ANOT) in eggs was developed and optimized. The samples were extracted using ASE with acetonitrile as the extractant and were purified by passage through a neutral alumina solid-phase extraction column. Then, the samples were analyzed using the GC-MS/MS method. The optimized method parameters were validated according to the requirements set forth by the European Union and the Food and Drug Administration. The average recoveries of dinitolmide and 3-ANOT from eggs (egg white, egg yolk, and whole egg) at the limit of quantification (LOQ), 0.5 maximum residue limit (MRL), 1 MRL, and 2 MRL were 82.74% to 87.49%, the relative standard deviations (RSDs) were less than 4.63%, and the intra-day RSDs and the inter-day RSDs were 2.96% to 5.21% and 3.94% to 6.34%, respectively. The limits of detection and the LOQ were 0.8 to 2.8 µg/kg and 3.0 to 10.0 µg/kg, respectively. The decision limits (CCα ) were 3001.69 to 3006.48 µg/kg, and the detection capabilities (CCß ) were 3001.74 to 3005.22 µg/kg. Finally, the new method was successfully applied to the quantitative determination of dinitolmide and 3-ANOT in 50 commercial eggs from local supermarkets.

Effect of dinitolmide intercalated into Montmorillonite on E. tenella infection in chickens.

To enhance the anti-coccidial effect of dinitolmide and reduce its residual, the dinitolmide/MMT compounds were synthesized by the method of solution intercalation via dinitolmide intercalated into Na + -montmorillonite (Na + -MMT). The structure of compounds was characterized by X-ray diffraction and Fourier transformed infrared. Its anti-coccidial effect was examined by Eimeria tenella infection experiment. One hundred fifty AA broiler chickens were divided into five groups. Chickens were orally inoculated with 5 × 10(4) E. tenella oocysts after dinitolmide was given. Their curative effects were observed. The results showed that intercalated dinitolmide expanded the basal spacing (d 001) of MMT from 12.6 to 15.2 Å. The IR bands of amide group in dinitolmide/MMT were detected at 1,533 cm(-1) which showed that dinitolmide was successfully intercalated into the interlayer spaces of MMT. The dinitolmide/MMT showed higher anti-coccidian oocyst activity compared with dinitolmide (p < 0.05). The dinitolmide/MMT compound can significantly increase body weight gains and reduce bloody diarrhea, lesion score, and oocyst excretion. The anti-coccidia index of dinitolmide/MMT group (165.21) is much higher than dinitolmide group (88.84). The dinitolmide/MMT hybrid systems can be more effective in control of coccidiosis in comparison to dinitolmide.

Depletion of the monoamino metabolites of Zoalene during frozen storage.

3-Amino-5-nitro-o-toluamide and 5-amino-3-nitro-o-toluamide, the principal metabolites in the tissues of chickens fed a diet containing Zoalene (3,5-dinitro-o-toluamide), were shown to deplete in frozen liver tissues stored up to 1 year at -20 degrees C, but not at -70 degrees C. A slight loss of 5-amino-3-nitro-o-toluamide also occurred in thigh muscle at -20 degrees C. Evidence is presented that demonstrates that the depletion of the metabolites was partially the result of their transformation to glucopyranosyl derivatives; both the alpha- and beta-anomers of the 5-amino-3-nitro-o-toluamide conjugate were observed in liquid chromatograms of tissue extracts.

Effects of different coccidiostats on performance of large white turkeys.

Two trials were conducted to evaluate the effects of feeding various anticoccidial products to turkeys to 8 wk and then growing to market age (16 wk for hens and 20 wk for toms). Anticoccidials evaluated in the first trial included amprolium at 187.5 mg/kg for 0 to 4 wk and 125 mg/kg for 4 to 8 wk; butynorate at 375 mg/kg for 0 to 8 wk; monensin at both 60 (MON-60) and 100 mg/kg for 0 to 8 wk; zoalene at 187.5 mg/kg for 0 to 4 wk and 125 mg/kg for 4 to 8 wk; and halofuginone at 3 mg/kg for 0 to 8 wk. In the second trial, MON-60 was replaced by a combination of sulfadimethoxine (62.5 mg/kg) plus ormetoprim (37.5 mg/kg) for 0 to 8 wk. In each trial each treatment was fed to four pens of 16 hens and four pens of 12 toms. Several of the anticoccidials significantly influenced the weight of both hens and toms by producing lower weights at the end of the 8-wk feeding period than birds in other treatments. However, after removal of the anticoccidials, compensatory gains were observed in almost every instance. Significant effects of previous anticoccidial feeding were noted on body weight of hens at 16 wk but not on weights of toms at 20 wk.

Efficacy of different anticoccidials against experimental coccidiosis in large white turkeys.

Two trials were conducted to compare the efficacy of currently approved anticoccidials for turkeys against challenge using a field isolate of mixed Eimeria species; E. adenoides, E. gallopavonis, and E. meleagrimitis. Poults in wire-floored cages were fed unmedicated diets from day-old to 3 wk of age. Diets were supplemented with either amprolium (AMP, 125 mg/kg), butynorate (BUT, 375 mg/kg), monensin (MON-60, 60 mg/kg; MON-100, 100 mg/kg), halofuginone (HAL; 3 mg/kg), zoalene (ZOA; 125 mg/kg), or sulfadimethoxine plus ormetoprim (SUL + ORM, 62.5 mg/kg and 37.5 mg/kg, respectively). After 2 days on the test diets, poults were individually weighed and inoculated with sporulated coccidial oocysts from the field isolate. Total fecal collections were obtained for Days 0 to 5 and 6 to 10 to estimate oocyst output. At 10 days postinoculation, the birds were individually weighed and killed to determine severity of intestinal lesions. The HAL and MON were most effective and AMP, ZOA, and SUL + ORM were least effective in maintaining weight and in reducing the severity of intestinal lesions. All the coccidiostats tested reduced oocyst passage, but poults fed HAL produced fewer oocysts. The results demonstrated differences in efficacy among anticoccidials with the more recently approved drugs providing the best protection against coccidiosis.

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.

Ultrastructural studies of the effects of amprolium and dinitolmide on Eimeria acervulina macrogametes.

Medication of chicks with 125 ppm amprolium or dinitolmide adversely affected oocyst sporulation of Eimeria acervulina (Weybridge strain). Dinitolmide delayed oocyst production and no oocyst wall formation was seen up to 168 h post infection. Both drugs caused large numbers of abnormally small wall-forming bodies to be produced in the macrogametes. In amprolium-fed chicks, abnormal oocyst wall formation was seen. It was concluded that the main drug action was against wall forming bodies of type 2.

Diseases diagnosed in replacement layer and breeder chicken flocks in Victoria, Australia, 1977 to 1985.

During the period from July 1977 to June 1985, specimens from 766 replacement layer and breeder chicken flocks were submitted to the Veterinary Research Institute, Victoria, Australia, for investigation of morbidity, mortality and, or, poor performance. A total of 5940 necropsies, as well as numerous ancillary tests, were performed. The findings for these flocks are presented and the consequences of some particular diseases noted.

[Poisoning in swine]. / Vergiftungen beim Schwein.

For clinical interests it is advisable to subdivide cases of swine poisoning in such as caused by food, drugs and environmental poisonings. This division gives pointers to aetiologic connections and special measures necessary for the clearing of the processes. With food poisoning mycotoxicoses play an evermore important role, whereas poisonings by trace elements are on the decrease. Sodium chloride poisoning often results primarily from insufficient water supply. With environmental poisonings carbon monoxide and cyanamide intoxication are presented. Poisonings caused by drugs are mainly the result of an overdose, of segregation in food or of non-licensed drugs. A relatively unknown swine poisoning by a drug against coccidiosis--licensed for poultry--is described.

Liquid chromatographic determination of zoalene and its metabolites in chicken tissues with electrochemical detection.

A procedure is described for the quantitation of Zoalene (3,5-dinitro-o-toluamide) and its 2 major monoamino metabolites in chicken tissues. The method includes blender extraction of tissue with chloroformethyl acetate (1 + 1), adsorption of the drug and metabolites on neutral alumina, and subsequent elution of the residues with pH 3.5 formate buffer-methanol (6.5 + 3.5). Recovered residues were separated on a 5 micron C18 column with the alumina eluting solvent as the LC mobile phase. The parent drug and metabolites were detected and quantitated with an electrochemical detector in the reductive mode with a minimum level of reliable measurement of 0.1 ppm. Overall mean recoveries greater than 85% were obtained with Zoalene and its 2 monoamino metabolites in breast, thigh, and liver tissues fortified with 0.25-2.00 ppm. The results on tissues from chickens fed a diet containing 0.0125% Zoalene are presented.

Liquid chromatographic determination of zoalene in medicated feeds.

A rapid, reliable separation and quantitation of zoalene (3,5-dinitro-o-toluamide) from feeds is accomplished by using reverse phase liquid chromatography (LC) and ultraviolet detection. An extraction technique which is similar to the present AOAC official colorimetric method is used before chromatographic analysis. This extraction is followed by an activated alumina cleanup and LC to separate zoalene from feed matrix. The methodology was applied to a variety of spiked feed matrices, and yielded good recoveries. Liquid chromatographic results were shown to correlate with colorimetric determinations.

Toxicity in utility pigeons caused by the coccidiostat dinitolmide.

Utility pigeons on 3 farms were affected by a severe neurological disturbance manifested by fine tremors, rolling gait and incoordination. Affected birds had necrosis of the Purkinje cells of the cerebellar cortex. The birds from the 3 farms had been fed a pelleted diet obtained from a single mix of feed prepared by a stockfeed manufacturer. When feed obtained from the farms was given to utility pigeons under controlled conditions at the laboratory, they suffered the same clinical signs and lesions as seen in birds from the farms. Cross-bred cockerels were not affected when given this feed from day-old to 6 weeks of age. Samples of the feed contained 185 to 226 ppm of the coccidiostat dinitolmide. Utility pigeons given a prepared diet containing a similar level (234 ppm) of dinitolmide developed an identical condition after 3 to 4 weeks feeding. Laying hens were not affected by this feed. Dimetridazole, which was also present in some of the original feed samples, did not exacerbate the condition when included in the diet at a rate of 233 ppm. Production of squabs from the affected farms was dramatically reduced, and many of the breeder had to be culled because of the long-standing neurological disease.

Anticoccidial drugs and duckling performance to four weeks of age.

Frequently, publications pertaining to waterfowl state that medicated feeds should not be fed to ducklings and goslings. In some localities, producers and hobbyists who raise a small number of ducklings and goslings can purchase only medicated chick, turkey, or gamebird starter and grower feeds. Because of the lack of documented information on this subject and the numerous requests for advice on this matter, anticoccidial drugs, zoalene, sulfaquinoxaline, and amprolium, were mixed in mash feed and fed to Khaki Campbell male ducklings to 4 weeks of age. No significant differences in mean body weight, mortality, and anatomical development were observed among the treatments. Medicated commercial crumble turkey and chick starter feeds produced significantly better feed conversion than the mash medicated or nonmedicated feeds. These differences can be attributed to greater feed spillage with the mash feed. Some ducklings in all treatments showed unsteadiness of gait and shaky legs. These conditions disappeared when the ducklings were moved from the battery brooder to an outside pen. Zoalene, sulfaquinoxaline, and amprolium used at the recommended levels for chickens and turkeys did not cause any leg or anatomical problems in ducklings.