A sequential procedure for rapid and accurate identification of putative trichomonacidal agents.

In the current report, a sequential step-wise methodology based on in silico, in vitro and in vivo experimental procedures for the prompt detection of potential trichomonacidal drugs is proposed. A combinatorial of 12 QSAR (Quantitative Structure-Activity Relationship) models based on Linear Discrimination Analysis (LDA) are suggested for the rational identification of new trichomonacidal drugs from virtual screening of in house chemical libraries and drug databases. Subsequently, compounds selected as potential anti-trichomonas are screened in vitro against Trichomonas vaginalis. Finally, molecules with specific trichomonacidal activity are evaluated in vivo. Herein, different molecules were exposed to the proposed methodology. Firstly, the agents were virtually screened and two of the eight molecules (G-1 and dimetridazole) were classified as trichomonacidals by the 12 models. Subsequently both drugs were proved in vitro and in vivo following the workflow procedure. Although a remarkable in vitro activity was observed in both cases, dimetridazole achieved higher MIC100 activity than metronidazole against the resistant isolate. Furthermore, the in vivo models showed a remarkable reduction of lesions of more than 55% in both compounds. These observations support the current flowchart screening and suggest the use of dimetridazole as a promising drug-like scaffold for novel therapeutic alternatives against T. vaginalis resistant infections.

In vivo effect of herbal products against Histomonas meleagridis in turkeys.

Histomoniasis is a serious disease in poultry. All chemotherapeutics with known efficacy against its causative agent, Histomonas meleagridis, have been banned from use as prophylactic or therapeutic use in production animals. In a search for possible alternatives, the in vivo effects of the herbal products Enteroguard and Protophyt were examined. Two-week-old turkeys allocated into 13 groups of 18 birds were either sham inoculated (negative control group) or were inoculated with 100, 3162 or 200 000 histomonads per bird. Control groups (no feed additives, dimetridazole, or Histostat-50) were included in the study. No morbidity or mortality was observed in the negative control group or in the groups inoculated with 100 histomonads per bird. Mortality was 100% in the groups inoculated with 200 000 histomonads per bird and either untreated (positive control group) or receiving Protophyt SP, Protophyt SP and Protophyt B, Enteroguard, or Histostat-50. Mortality was 17% in the dimetridazole-treated group. In the groups inoculated with 3162 histomonads per bird, mortality was 100% for the positive control group and the group receiving Enteroguard, and was 94% in the group receiving Protophyt SP. In the present study, Enteroguard or Protophyt was not found to be effective against histomoniasis.

In vitro activity of therapeutic drugs against Histomonas meleagridis (Smith, 1895).

Histomoniasis or blackhead is a life-threatening disease of turkeys that is caused by a flagellated protozoan, Histomonas meleagridis. The development of an assay to measure the sensitivity of drugs traditionally used against this parasite, as reputed to be effective against other protozoan parasites, is described. The in vitro minimum lethal concentrations (MLC), time for drug efficacy, and parasite viability after removal of residual drugs were determined. Three of the 10 tested drugs, fenbendazole, albendazole, and sulfadiazine, were found to be ineffective against H. meleagridis. Nifursol, the only compound still authorized as a feed additive in Europe, is an inhibiting agent but is not lethal in vitro. Roxarsone, an arsenical derivate similar to nitarsone (the only authorized drug in United States), is effective at high concentration (200 microg/mL) after a long exposure (48 h). The lethal activity of dimetridazole, metronidazole, ronidazole, tinidazole, and furazolidone in vitro was demonstrated. Dimetridazole (MLC = 25 microg/mL after 6 h of exposure), metronidazole (MLC = 50 microg/mL after 24 h), and furazolidone (MLC = 50 microg/mL after 24 h) are rapidly effective at low concentrations. These results confirm the effectiveness of dimetridazole, a drug that has been used in the treatment and prevention of blackhead. In May 2002 this compound was removed as feed additive in Europe.

Efficacy of various chemotherapeutic agents on the growth of Spironucleus vortens, an intestinal parasite of the freshwater angelfish.

Seven chemotherapeutic agents (dimetridazole, metronidazole, pyrimethamine, albendazole, fenbendazole, mebendazole and magnesium sulfate) were examined for growth inhibition on the cultivation of Spironucleus vortens. Dimetridazole and metronidazole were effective in inhibiting the parasite's growth. At concentrations of 1 microgram ml-1 or higher, both dramatically decreased numbers of parasites. At 24 h exposure, 33% of parasites were inhibited when exposed to dimetridazole or metronidazole at concentrations of 2 and 4 micrograms ml-1, respectively. Dimetridazole at 4 micrograms ml-1 or higher concentrations decreased the number of organisms to 50% or less after 48 h exposure. During the same period of time, the numbers of parasites decreased to 50% or less when exposed to metronidazole at 6 micrograms ml-1 or higher. Pyrimethamine at concentrations of 1 to 10 micrograms ml-1 was not effective in inhibiting the parasite's growth. Albendazole and fenbendazole at concentrations of 0.1 and 0.5 microgram ml-1 were similar in inhibiting the growth of the organism. Both compounds suppressed parasite growth at concentrations of 1.0 microgram ml-1 or higher after 24 h exposure. Mebendazole inhibited the parasite's growth at concentrations of 0.5 microgram ml-1 or higher. At 72 h exposure, 45 to 50% of the parasites were inhibited when exposed to mebendazole at concentrations higher than 0.5 microgram ml-1. Magnesium sulfate at concentrations of 70 mg ml-1 or higher also suppressed the growth of parasites after 24 h exposure. These results indicate that dimetridazole, metronidazole and mebendazole are the most effective chemotherapeutic agents in vitro at inhibiting the growth of S. vortens.

Evaluation of the genotoxic activity of metronidazole and dimetridazole in human lymphocytes by the comet assay.

The genotoxicity of metronidazole (MZ) and dimetridazole (DZ) has been evaluated in human lymphocytes using the comet assay. The test has been performed using 3 doses (58.4, 175.2 and 292.1 microM for MZ; and 70.9, 212.6 and 354.3 microM for DZ) under 3 experimental protocols: aerobiosis, anaerobiosis (90% N2, 10% CO2) and with the presence of the microsomal fraction S9 mix. The effects of 4 antioxidants (8-hydroxyquinoline (8HQ), vitamin C (VitC), catalase (CAT) and superoxide dismutase (SOD), have been investigated on DNA damage generated by fixed concentrations of MZ (292.1 microM) and DZ (354.4 microM). In aerobic conditions, MZ and DZ produced significant dose-response relationships. The dose-related effects of both drugs decreased or were abolished in anaerobic conditions or in presence of S9 mix. 8HQ, VitC, CAT and SOD induced dose-related protective responses against DNA damage due to MZ and DZ. These findings suggest that MZ and DZ induce DNA damage in human lymphocytes through the futile cycle. The one-electron reduction of the drugs leads to the production of nitro radical anions. In the presence of oxygen, these radicals are reoxidized and generate oxygen-activated species.

Sensitivity of strains of Serpulina hyodysenteriae isolated in Hungary to chemotherapeutic drugs.

The sensitivity of 332 strains of Serpulina hyodysenteriae isolated in Hungary between 1978 and 1992 was tested against seven chemotherapeutic drugs frequently used for the treatment of swine dysentery, and the changes in the patterns of resistance were also monitored. All the strains remained sensitive to carbadox, with minimum inhibitory concentrations (MIC) of only 0.05 to 0.40 microgram/ml at present. The susceptibility of the strains to dimetridazole has gradually decreased, but about half of the strains are still sensitive, with large numbers of "moderately sensitive' strains; the MIC values varied within wide limits (0.1 to 50 micrograms/ml). Most of the strains were resistant to tylosin, with MIC values from 0.1 to 100 micrograms/ml. The number of strains resistant to lincomycin has gradually increased, but about half of the strains remain sensitive; the MIC values ranged from 0.2 to 100 micrograms/ml. Recently, tiamulin has proved the most effective antibiotic, but some resistant strains have already emerged (MIC values 0.05 to 50 micrograms/ml). Monensin was good for the prevention of swine dysentery, but resistance may evolve quickly; the MIC values ranged from 0.4 to 25 micrograms/ml. For sedecamycin, the MIC values (6.25 to 100 micrograms/ml) were much higher than expected.

Antimicrobial susceptibility testing of Serpulina hyodysenteriae.

The macrobroth dilution technique was used to test the in-vitro effectiveness of 4 commonly used antimicrobial agents against 23 Australian isolates and 7 overseas strains of Serpulina hyodysenteriae. Minimum inhibitory concentrations and minimum bactericidal concentrations were determined. The growth of 90% of isolates was inhibited by dimetridazole at a concentration of 4 micrograms/mL, and by tiamulin at 8 micrograms/mL. Australian isolates resistant to both antimicrobial agents were identified. Lincomycin was less effective than these antimicrobial agents, with 90% of isolates requiring a concentration of 128 micrograms/mL for inhibition of growth, and 54% being susceptible at 64 micrograms/mL. Tylosin did not prevent the growth of the majority of S hyodysenteriae isolates tested, and 90% were resistant to concentrations of > or = 128 micrograms/mL. Resistant isolates came from different geographical areas. Resistance was not related to overall genetic background of the spirochaetes, and was not correlated with the presence of plasmids or the serogroup of the isolates.

In vitro nitroimidazole resistance of Trichomonas gallinae and successful therapy with an increased dosage of ronidazole in racing pigeons (Columba livia domestica).

Six out of eight different Trichomonas gallinae strains isolated from racing pigeons proved to be resistant to the nitroimidazole drugs ronidazole, carnidazole and metronidazole. The minimal cytocidal concentration of ronidazole was determined in in vitro experiments. Moreover, a therapeutic dose for ronidazole was determined for the control of trichomoniasis in pigeons from which the resistant T. gallinae strains were isolated. It was a 5-fold increase of the recommended ronidazole dosage which eliminated the infection in affected pigeons.

Minimal inhibitory concentrations of five antimicrobials against Treponema hyodysenteriae and Treponema innocens.

The minimal inhibitory concentrations of carbadox, dimetridazole, lincomycin, ronidazole, and tiamulin against isolates of Treponema hyodysenteriae and Treponema innocens were determined by an agar-dilution method. The results obtained indicated that tiamulin was the most effective antimicrobial in vitro against T. hyodysenteriae, followed by carbadox. Dimetridazole, lincomycin, and ronidazole had poor efficacy in vitro against the T. hyodysenteriae isolates. Isolates of T. innocens were more sensitive to the various antimicrobials. Carbadox and tiamulin were the most effective in vitro, followed by ronidazole, dimetridazole, and lincomycin.

Effect of intestinal flagellate Spironucleus (Hexamita) muris and of dimetridazole on intestinal microflora in thymus-defficient (nude) mice.

Two groups of the intestinal microflora, the lactobacilli and the coliforms, were examined in thymus-deficient (nude) mice during the development of an experimental infection with the intestinal flagellate Spironucleus (Hexamita) muris and during the treatment with dimetridazole. The observed significant decrease in the number of lactobacilli under infection was probably due to the fact that the protozoan parasite fed on the microbes. Dimetridazole (0.3% in drinking water) did not influence the quantity of the lactobacilli but, owing to its selective killing of anaerobes and the lack of their antagonistic activity, a 100- to 1000-fold rise in the number of coliform microbes was observed. No of the drugs tested (dimetridazole, ornidazole, metronidazole, tinidazole, carbimazole BP and chlormethoxy-acridilamino-diethylamino-propanol-dihydrochliorde) was fully successful in the treatment of experimental spironucleosis in mice (Kunstýr, 1978) and it is suggested that recent reports on the therapeutic success of tinidazole in human giardiasis be treated with caution.

Influence of cellular environment on toxicity of nitroheterocycles.

The preferential sensitivity of hypoxic cells to nitroheteroxycles is thought to result from the actions of toxic intermediates of drug reduction produced under hypoxic conditions. However, a lack of oxygen also alters the biochemical state of the cell and may indirectly enhance the sensitivity, of hypoxic cells to these drugs. This hypothesis was tested by 'conditioning' mouse L-929 cells in oxygen-free buffer, then exposing the cells to nitrofurazone under both aerobic and anaerobic conditions. After conditioning, the rate of cell inactivation by nitrofurazone was equal in air or nitrogen-equilibrated buffer. Pretreatment of cells in 1 muM rotenone or 0.5 mM 2,4-dinitrophenol for one hour under aerobic conditions increased the sensitivity of the cells to nitrofurazone under aerobic conditions. Similar rates of cell killing were obtained when mouse L-cells were heated in buffer for 30 min at 43 degrees before incubation with nitrofurazone in either air or nitrogen. Also, incubation of cells with nitrofurazone in the presence of 0.1% glucose, or at a cell density less than 10(5) cells/ml significantly enhanced cell killing, especially under aerobic conditions. Thus, the intracellular state of the cell, manipulated by altering the cellular environment, influenced the cellular sensitivity to nitrofurazone. Similar results were not, however, obtained with the nitroimidazoles, dimetronidazole and misonidazole; pretreatment for 2 h in buffer under anaerobic conditions did not increase the sensitivity of L cells to subsequent drug treatment in air-equilibrated buffer.

Sensitivity in vitro to dimetridazole of treponemes associated with swine dysentery.

The minimum inhibitory concentration (MIC) of dimetridazole (DMZ) against Treponema hyodysenteriae (55 isolates) obtained over the period 1974-77 from individual pigs with swine dysentery from 41 herds where DMZ had been in use was determined. The MIC was less than or equal to 5.0 microgram per ml for 54 of the isolates and differences in the distribution of MICs between the annual sampling periods were not significant (P less than 0.05). There was no decrease in sensitivity of T hyodysenteriae to DMZ during the survey.

Selective decontamination of the digestive tract of Syrian hamsters.

Conventional Syrian hamsters colonized with aerobic gram-negative bacteria such as Pasteurella pneumotropica and various Enterobacteriaceae species were successfully and permanently freed from these microorganisms by oral treatment for 4 weeks with dihydrostreptomycin and 'Orabase' premixed with appropriate antibiotics. Concomitant oral treatment with dimetridazol for the elimination of intestinal flagellates was unsuccessful. During treatment the animals were maintained under germ-free isolation conditions.

Resistance of faecal cysts of Spironucleus muris to some physical factors and chemical substances.

The effect of 5 disinfectants, and of saturated zinc chloride solution + sodium chloride solution, 2.5% potassium dichromate solution, and 0.12% dimetridazole on faecal cysts of Spironucleus muris were tested in vitro. The resistance of the cysts to high and low temperatures, low pH, high osmotic pressure, centrifugation and desiccation was also tested. After treatment the morphology of the cysts was observed microscopically and their infectivity tested in vivo on sensitive thymus-deficient nude mice. The cysts ceased to be infective after treatment with most of usual disinfectants and by high temperature (45 degrees C for 30 min). They resisted 0.12% dimetridazole, low temperature (-196 degrees C), low pH (2.2), high osmotic pressure (distilled water and 30% 'Ficoll'), centrifugation (1500 g/20 min) and desiccation (room temperature for 14 days). These data may be useful for the control of Spironucleus muris infection in rodents and for cryopreservation of the parasite for experimental purposes.

The effect of antimicrobial feed additives on the colonization of the alimentary tract of chickens by Salmonella typhimurium.

Groups of 33 chickens were fed continuously on diets containing feed additives that are employed commercially for a variety of purposes, and were infected orally when 4 days old with a nalidixic acid-resistant mutant of Salmonella typhimurium. The amount of S. typhimurium organisms excreted in their faeces was estimated by culturing them at intervals and in a standard manner on brilliant green agar containing sodium nalidixate; when the chickens were killed their caecal contents were examined by the same technique.Avoparcin and lincomycin, like nitrovin and tylosin (Smith & Tucker, 1975b), favoured colonization of the alimentary tract by the S. typhimurium organisms as shown by the fact that the chickens to which they were fed excreted these organisms in their faeces in higher concentration and for longer periods of time than did chickens fed on non-medicated diets. Amprolium, monensin, dimetridazole, arsenilic acid and nitro-hydroxyphenylarsonate had no obvious effect on the salmonella excretion pattern.When only five chickens in each group were experimentally infected so that the effect of the feed additives on infections acquired by contact could be monitored, avoparcin, lincomycin, nitrovin and tylosin again favoured colonization of the alimentary tract with the S. typhimurium organisms and so did dimetridazole. Arsenilic acid, in contrast, hindered the development of infection. Amprolium, monensin and nitro-hydroxyphenylarsonate were without obvious effect.Many of the chickens that were fed on diets that favoured S. typhimurium colonization, but not those fed on non-medicated diets, were still excreting S. typhimurium organisms in their faeces when they were killed at 56 days of age, the age at which broiler chickens kept under commercial conditions are usually slaughtered.