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.

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.

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.

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.

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.

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.

Effect of amprolium and dinitolmide on sporulation of oocysts of field isolates of Eimeria acervulina.

A laboratory strain of Eimeria acervulina and 9 field isolates consisting principally of E. acervulina were tested for sensitivity to amprolium (125 p.p.m.) or dinitolmide (125 p.p.m.) in the food and for effects of the drugs on sporulation of oocysts. Judged by weight gains and lesion scores, medicaments were only partially effective against the 9 field isolates, but were highly effective against the laboratory strain. Oocysts were produced in all the infections but the percentage sporulation of oocysts from field isolates was much higher than sporulation of oocysts of the "drug sensitive' laboratory strain. These results show that coccidia that are resistant to either amprolium or dinitolmide are able to cause lesions in the presence of the drugs and the oocysts that are produced will sporulate normally.

Ultrastructural changes in the macrogamete and early oocyst of Eimeria maxima resulting from drug treatment.

The ultrastructure of the macrogamete and developing oocyst of Eimeria maxima (Weybridge strain) was examined in the intestinal cells of chicks fed 3 different anticoccidial drugs. Amprolium at 125 p.p.m., arprinocid at 35 p.p.m. and dinitolmide at 250 p.p.m. caused considerable morphological abnormality and incomplete development of the wall-forming bodies of Type 2 (WFB II), which did not appear able to participate in oocyst wall formation. The wall-forming bodies of Type 1 (WFB I) were able in each case to participate in oocyst wall formation although amprolium and dinitolmide produced morphological abnormalities in them. In birds medicated with dinitolmide, the outer layer of the oocyst wall was formed initially at opposite poles of the macrogametes rather than as a uniform layer. Other abnormalities resulting from drug treatment are reported and some evidence that intravacuolar tubules may be formed by the parasite pellicle is presented.

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.

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.

The anticoccidial effects of amprolium, dinitolmide and monensin against Eimeria maxima, E, brunetti and E. acervulina with particular reference to oocyst sporulation.

Chicks infected with the Weybridge strains of Eimeria maxima and E. acervulina were not protected by the normal levels of amprolium, but the sporulation of the oocysts was inhibited. With higher drug concentrations, fewer oocysts were produced. E brunetti was not so markedly affected, although oocyst sporulation was reduced by the higher dosage levels. The effects were not enhanced by the inclusion of ethopabate. With dinitolmide the phenomenon was not so marked, although oocysts of E. maxima and E. acervulina were reduced in numbers by the normal drug concentration and sporulation was reduced when this level was increased. Much higher drug levels were required to obtain these effects with E. brunetti. Monensin at 120 ppm affected neither oocyst numbers nor their sporulation in any of the species tested. The significance of the effects of anticoccidial drugs on gametogony and sporogony is discussed.

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.

Sequential use of coccidiostats: effect on development by Eimeria tenella of resistance to amprolium, nicarbazin, Unistat, and zoalene.

A strain of the cecal coccidian of chickens, Eimeria tenella, was propagated serially in chickens fed mash containing amprolium, nicarbazin, Unistat, or zoalene. Each group of chickens received a different coccidiostat on a rotating basis. The strain was propagated through 40 groups of chickens; thus, the strain was intermittently exposed 10 times to each coccidiostat. The end product of this simulated shuttle program of prophylactic anticoccidial medication was a strain resistant to three of the four coccidiostats involved. Resistance to nicarbazin was not evident.

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.