Methyl Benzoquate Research Articles

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.

Studies of the mitochondria from Eimeria tenella and inhibition of the electron transport by quinolone coccidiostats

Intact but fragile mitochondria were isolated from unsporulated oocysts of Eimeria tenella. The mitochondria respired in response to succinate, malate plus pyruvate, and l-ascorbate at rates of 1.00, 0.40, and 0.25 μl 0 2/min/mg protein, respectively. Spectrophotometric analyses of the cytochromes in mitochondria and whole oocysts revealed b-type and o-type cytochromes, at roughly similar levels, but no cytochrome c could be detected. The mitochondrial respiration was inhibited by cyanide, azide, carbon monoxide, antimycin A, and 2-heptyl-4-hydroxyquinoline- N-oxide, but was relatively resistant to rotenone and amytal. The quinolone coccidiostats buquinolate, amquinate, methyl benzoquate, and decoquinate were identified as very powerful inhibitors of succinate and malate plus pyruvate supported respiration in E. tenella mitochondria. None of these four drugs exhibited any inhibitory effect on chicken liver mitochondria. Only 3 pmol of the quinolones per mg mitochondrial protein was needed to achieve 50% inhibition. The inhibition could not be reversed by coenzymes Q 6 or Q 10. Since the quinolones did not affect l-ascorbate-supported respiration or the activities of submitochondrial succinate dehydrogenase and NADH dehydrogenase, the site of action of the quinolone coccidiostats was tentatively identified as probably near cytochrome b in E. tenella mitochondria. Mitochondria isolated from an E. tenella amquinate-resistant mutant were much less susceptible to quinolone coccidiostats; 50% inhibition was attained by 300 pmol of the drugs/mg mitochondrial protein. The results suggest that the mechanism of action of quinolone coccidiostats is by inhibiting the cytochrome-mediated electron transport in the mitochondria of coccidia. 2-Hydroxynaphthoquinone coccidiostats were identified as inhibitors of mitochondrial respiration of both E. tenella and chicken liver. They inhibited submitochondrial succinate dehydrogenase and NADH dehydrogenase of E. tenella, and remained equally active against the mitochondrial function of E. tenella amquinolate-resistant mutant.

Eimeria tenella in chickens: development of resistance to quinolone anticoccidial drugs.

The development of drug resistance by the present Houghton strain of Eimeria tenella to the quinolones, methyl benzoquate and buquinolate, was found to take place after a single experimental passage. The development of resistance was independent of drug selection pressure and showed cross resistance to other quinolones, but not to amprolium and robenidine. When the Weybridge, Beltsville and Elberfeld strains of E. tenella were compared under similar laboratory conditions, the Weybridge and Elberfeld strains developed resistance to methyl benzoquate after 6 passages and the Beltsville after 5. Studies on the response of the Houghton strain to methyl benzoquate and buquinolate revealed that the drugs did not completely control the infection as measured by weight gain and that oocyst production was not suppressed. These observations indicate that the strain had already acquired some resistance to these drugs. This was confirmed by examining the resistance to methyl benzoquate of a culture of the Houghton strain of E. tenella which had been stored frozen in liquid nitrogen since 1969. This showed full sensitivity to the drug and developed resistance after 8 passages. This suggests that drug tolerance has been acquired by the Houghton strain since 1969. Oocyst lines were established from the Houghton strain by infecting single birds with approximately 10 oocysts. Eleven of these lines were found to be sensitive to methyl benzoquate, and nine to give rise to resistant parasites. It is concluded that the Houghton strain is contaminated by a small number of resistant oocysts which can be eliminated from a culture by dilution of the challenge inoculum. One of these Houghton oocyst lines, sensitive to methyl benzoquate, developed resistance after 8 serial passages.

Lerbek, a synergistic mixture of methyl benzoquate and clopidol for the prevention of chicken coccidiosis.

Methyl benzoquate at 20 ppm or clopidol at 250 ppm in the diet show true coccidiostatic activity in infections with Eimeria tenella in the chick. Lerbek, a synergistic mixture yielding 8-35 ppm methyl benzoquate and 100 ppm clopidol, produced a partially coccidiocidal effect; relapses following restricted periods of medication were less severe, and suppression of parasite development following delayed medication was quicker and more effective with either component of the mixture used alone.

Use of chick embryo infections for the study of drug resistance in Eimeria tenella.

The activity of 12 anticoccidial drugs against an embryo-adapted strain of Eimeria tenella has been investigated. After seven and five serial passages respectively, in the presence of drug, resistance was developed to methyl benzoquate and buquinolate. A strain of E. tenella made resistant by exposure to drugs in chickens was resistant to the same drugs when evaluated in infections of chick embryos. This relatively simple technique would appear to offer advantages over existing methods for the study of drug resistance.

Eimeria tenella: Anticoccidial drug activity in cell cultures

Anticoccidial activity of 16 drugs against Eimeria tenella was studied in cell cultures. Compounds active at 100 μg/ml or less were ethopabate, amprolium, monensin, nicarbazin, glycarbylamide, robenidine, zoalene, sulfamethazine, nitrofurazone, decoquinate, clopidol, aklomide, buquinolate, methyl benzoquate, and nitromide. Roxarsone was not active at 100 μg/ml. One level of each of nine compounds was selected for addition-withdrawal studies with suppression of development to second generation schizonts 96 hr postinoculation of sporozoites used as an endpoint of activity. Drugs were added to cultures 24, 48 or 72 hr or were added at 0 hr and withdrawn 24, 48 or 72 hr after introduction of sporozoites. Clopidol, zoalene, sulfamethazine, glycarbylamide, and nicarbazin delayed development, roughly equivalent to the time the compound was in the culture, even when compounds were withdrawn at 24 or 48 hr. Withdrawal of nicarbazin, glycarbylamide, and zoalene 72 hr after inoculation of sporozoites allowed some development of small immature schizonts of the first generation. A delay in addition of anti-coccidials until 24 hr did not affect the observed endpoint with any compounds except clopidol and glycarbylamide. When compounds were added 48 hr postinoculation of sporozoites, the activity of amprolium, glycarbylamide, sulfamethazine, and zoalene was not detected. Only with monensin was activity demonstrable when added at 72 hr. This study demonstrated that in vitro techniques can be used not only to titrate the activity of anticoccidial drugs and identify the stage of development where the drug is most active, but to determine coccidiocidal versus coccidiostatic actvity as well.

Comparative Studies with Anticoccidials and Three Species of Chicken Coccidia In vivo and In vitro

At practicable use levels in chickens, some coccidiostats exhibit activity against but 1 or a few species of coccidia, while others have wide-spectrum activity. This might be due to a true variation in species response, or might be a reflection of differences in metabolism and distribution of different drugs in the various sites parasitized by the several species of coccidia. Activities of a number of drugs against Eimeria brunetti, E. necatrix, and E. tenella have been compared both in the chick and in tissue culture. These studies indicate a true species variation in sensitivity to a number of growth factor antagonists, and in the cases of zoalene and methyl benzoquate, possible modifications in activity by drug metabolism and/or distribution. Anticoccidials of the 1940's and 1950's were restricted in the spectrum of their activity at economically and toxicologically usable levels. Thus the sulphonamides showed considerably more activity against intestinal species of coccidia than against Eimeria tenella, while others such as dinitrobenzamide and amprolium were more active against E. tenella than against intestinal species. Wide-spectrum activity was achieved initially by using mixtures of drugs (e.g., Unistat, Pancoxin, Darvisul, Whitsyn). Subsequently, with the discovery of the quinolone, pyridone, and guanidine coccidiostats (e.g., methyl benzoquate, meticlorpindol, robenidine), wide-spectrum control at practical levels could be achieved with a single compound. The differing responses of the various species to different drugs in the chicken gut might be due to genuine species differences in susceptibility, but it could well be merely a reflection of drug absorption, metabolism, and distribution interacting with the differing locations of the various species in the gut. The development of tissue culture techniques for the in vitro cultivation of several species of coccidia has enabled us to compare the anticoccidial activities of a number of drugs against E. tenella, E. necatrix, and E. brunetti without the complicating variables of drug absorption and metabolism and parasite location.

Maternal transfer of anticoccidial drugs in the chicken

Intramuscular inoculation of pullets with large amounts of methyl-7-benzyloxy-6-butyl-1, d-dihydro-4-oxquinoline-3-carboxylate (Methyl benzoquate) 3, 5-dichloro-2, 6-dimethyl-4-pyridinol (Clopidol) or 1, 3, -bis (parachlorobenzylidene-amino-guanidine hydrochloride (Robenzidene) resulted in the transfer of sufficient drug to protect embryos against E. tenella from eggs laid by hens treated up to 70 days previously, but afforded little or no protection to chicks hatched from eggs produced by hens given these drugs. Inoculation of 5 mg. Robenzidene by the yold sac of 7-day-old chick embryos protected 3-day-old chicks, obtained from these eggs. Concentrations of methyl benzoquate in the eggs of pullets given this drug in their diet at 0·002 per cent. were sufficient to protect 11-day-old embryos. The results indicate that laying hens used as a source of embryos or chicks for coccidiosis stduies should not be given anticoccidial drugs in their food and effective levels of anticoccidial agents remain for long periods in hens given the drugs intramuscularly.

Observations on a Drug Resistant Strain of Eimeria tenella

A strain of Eimeria tenella from the field was found to be resistant to the quinoline group of coccidiostats. There were no significant differences in mortality or microhaematocrit levels in chickens treated with methyl benzoquate at 0.005% and 0.025% as compared with untreated birds infected with this strain. In comparative experiments using this strain of E. tenella, deccoquinate did not control mortality or haemorrhage, clopidol prevented mortality but not haemorrhage, and robenzidene prevented both mortality and haemorrhage.

A Technique for Demonstration of Coccidiostatic Activity of Anticoccidial Agents

A new combination of parameters used in anticoccidial evaluation has been combined in a graphic representation to separate from coccidio- cidal drug activity. The procedure involves plotting graphically daily oocyst counts and daily lesion scores while birds are on medication, during the period following artificial inoculation, and during a period following drug withdrawal 10 days post- infection. Coccidiostatic activity is indicated by peaks in lesion scores and oocyst production following drug withdrawal. Coccidiocidal activity is indicated by absence of peaks during the withdrawal period combined with suppression of oocysts and lesion scores compared to non-medicated controls during the primary infection. In action against Eimeria tenella drugs were classified as follows: strongly coccidio- static-buquinolate, clopidol, decoquinate, methyl benzoquate, and anticoccidial C; coccidiocidal-chlorotetracycline, oxytetracycline, sulfachloropyrazine, sulfa- dimethoxine, sulfamethazine, sulfaquinoxaline, Tri-sulfa-lyte?, and tylosin; both coc- cidiostatic and coccidiocidal-amprolium plus ethopabate, anticoccidial A, monen- sin, nicarbazin, Novastat?, Polystat', RofenaidTM Unistat?, Whitsyn?-S, and zoalene. Since the activity of sulfanilamide was first demonstrated (Levine, 1939), an extensive volume of information has been published compar- ing the efficacy of the numerous anticoccidial drugs. The manufacture of these products has become a major pharmaceutical industry. Their use in preventive medication has been almost universally adopted by the poultry industry and it is now difficult to purchase non-medicated starter feed in the USA. Prevention of these several closely related coccidial diseases has been a major factor in per- mitting development of the gigantic broiler industry (Reid, 1968). In all of this development, the term coccidiostatic has been used although it is generally recognized that many of the more than 20 pharmaceutical products commercially used are primarily coccidiocidal rather than in their activity. A more accurate term would be anticoccidial agent but the term coccidiostat is so widely adopted that a change seems unlikely. Within the past three years several new anticoccidial drugs have been made available which are much more truly in their activity than those ABSTRACT: A new combination of parameters used in anticoccidial evaluation has been combined in a graphic representation to separate from coccidio- cidal drug activity. The procedure involves plotting graphically daily oocyst counts and daily lesion scores while birds are on medication, during the period following artificial inoculation, and during a period following drug withdrawal 10 days post- infection. Coccidiostatic activity is indicated by peaks in lesion scores and oocyst production following drug withdrawal. Coccidiocidal activity is indicated by absence of peaks during the withdrawal period combined with suppression of oocysts and lesion scores compared to non-medicated controls during the primary infection. In action against Eimeria tenella drugs were classified as follows: strongly coccidio- static-buquinolate, clopidol, decoquinate, methyl benzoquate, and anticoccidial C; coccidiocidal-chlorotetracycline, oxytetracycline, sulfachloropyrazine, sulfa- dimethoxine, sulfamethazine, sulfaquinoxaline, Tri-sulfa-lyte?, and tylosin; both coc- cidiostatic and coccidiocidal-amprolium plus ethopabate, anticoccidial A, monen- sin, nicarbazin, Novastat?, Polystat', RofenaidTM Unistat?, Whitsyn?-S, and zoalene. Since the activity of sulfanilamide was first demonstrated (Levine, 1939), an extensive volume of information has been published compar- ing the efficacy of the numerous anticoccidial drugs. The manufacture of these products has become a major pharmaceutical industry. Their use in preventive medication has been almost universally adopted by the poultry industry and it is now difficult to purchase non-medicated starter feed in the USA. Prevention of these several closely related coccidial diseases has been a major factor in per- mitting development of the gigantic broiler industry (Reid, 1968). In all of this development, the term coccidiostatic has been used although it is generally recognized that many of the more than 20 pharmaceutical products commercially used are primarily coccidiocidal rather than in their activity. A more accurate term would be anticoccidial agent but the term coccidiostat is so widely adopted that a change seems unlikely. Within the past three years several new anticoccidial drugs have been made available which are much more truly in their activity than those

The detection of prophylactic drugs in animal feeding stuffs by thin-layer chromatography.

A procedure is described for detecting the presence of seventeen prophylactic drugs commonly present in animal feeding stuffs. Only two solvent systems are used for separation purposes. The drugs investigated were: acinitrazole, 2-chloro-4-nitrobenzamide (aklomide), aminonitrothiazole, amprolium, N4-acetyl-4′-nitrosulphanilanilide (APNPS), buquinolate, decoquinate, dimetridazole, 3,5-dinitrobenzamide (DNBA), ethopabate, furazolidone, clopidol (metichlorpindol), nitrofurazone, pyrimethamine, methyl benzoquate, sulphaquinoxaline and 3,5-dinitro-o-toluamide (zoalene).

Eimeria: Effect of meticlorpindol and methyl benzoquate on endogenous stages in the chicken

Meticlorpindol and methyl benzoquate had no action on the sporozoites of Eimeria brunetti in vitro. The drugs exert an inhibitory action on the sporozoites after invasion of the intestinal mucosa when the sporozoites have completed their migration. Some drug must be absorbed from the intestine since chicks treated with meticlorpindol in the food were protected from an infection induced via the blood-stream. The drugs may have an effect on later stages of the life cycle of some species of Eimeria, but not with others. Sporozoites of E. tenella, E. mivati and E. acervulina could develop even after treatment for 60 days with meticlorpindol, whereas other species ( E. maxima) did not develop after 21 days of exposure to the drug. Possible reasons for the differences in survival of sporozoites of different species are discussed in relation to the turnover of intestinal epithelial cells and to the role of macrophages in the transport of sporozoites within the intestinal tissues. Chickens treated with meticlorpindol for 70 days did not exhibit latent infection after drug withdrawal, but were susceptible to infection with species of Eimeria used initially. This indicates that the presence of inhibited sporozoites did not stimulate resistance to reinfection.

Studies on the Mode of Action of Quinolone and Pyridone Coccidiostats

The primary action of the anticoccidial drugs methyl benzoquate, buquinolate, and meticlorpindol is to prevent the growth of, rather than to kill, coccidia. In the case of Eimeria tenella in the chick, only the sporozoite when it has first invaded the host cell is susceptible to this action, but this is due not to lack of susceptibility of later stages, but rather to failure of the drug to penetrate deep into the gut wall. Later stages of E. brunetti are susceptible to methyl benzoquate given in the food as are later stages of E. tenella if the drug is given intravenously or the experiments are carried out in the chick embryo. Methyl benzoquate given intravenously in the chick forms a depot, mainly in the lung, which is able to protect against lethal coccidial challenges for several weeks. No success has been achieved in explaining the mode of action of the drugs at a molecular level, although the observations would be consistent with an inhibition of nucleic acid synthesis. The earliest compounds found to have marked anticoccidial activity were the sulphonamides, and as with bacteria, it has been shown that these act as competitive inhibitors of p-aminobenzoic acid (Horton-Smith and Boyland, 1946; Waletzky and Hughes, 1946). During studies involving treatment with varying concentrations of sulphadimidine over restricted parts of the life cycle of Eimeria tenella, it was shown (Kendall and McCullough, 1952; Davies and Kendall, 1954a, b) that the stages of the parasite present 72 to 96 hr after infection were most sensitive to the drug, whereas those present during the first 24 hr of the infection were only influenced by very high concentrations of sulphonamide. Moreover, although second generation schizonts appeared to be killed by sulphadimidine, early treatment of infections led only to suppression of the parasite, which was able to continue its development if the drug were later withdrawn. Potentiation of sulphonamides with substances such as pyrimethamine or diaveridine rely on the ability of the potentiator to interfere with the folic acid pathway in the parasite (Joyner and Kenldall, 1956). Amprolium was developed as a coccidiostat during a program of work on thiamine antagonists, and its anticoccidial activity can be annulled by thiamine in the chick (Rogers et al., 1960; Rogers, 1962) or in the chick embryo (Ryley, 1968). The present studies were undertaken in an attempt to throw some light on the mode of action of Received for publication 28 July 1967. the newly introduced quinolone coccidiostats methyl benzoquate (I.C.I. 55,052) (Bowie et al., 1967; Ryley, 1967) and buquinolate (Spencer et al., 1966) and the pyridone, meticlorpindol (Stock et al., 1967).

Methyl Benzoquate , a New Wide-Spectrum Coccidiostat for Chickens

Methyl benzoquate (7-benzyloxy-6-n-butyl-3-methoxycarbonyl quinol-4-one) administered continuously at a level of 0.001 per cent in the food gives excellent protection against Eimeria acervulina, E. brunetti, E. maxima, E. mivati, E. necatrix and E. tenella in chickens as judged by control of mortality, weight gain and oocyst output. It thus displays more than ten times the intrinsic activity of other available coccidiostats, and has a wider spectrum of activity than most. Le benzoquate de méthyle (7-benzyloxy-6-n-butyl-3-méthoxycarbonyl quinol-4-one), administré sans interruption au taux de 0,001 pour cent de nourriture, a engendré une excellente protection contre Eimeria acervulina, E. brunetti, E. maxima, E. mivati, E. necatrix et E. tenella chez les poulets, ainsi qu’il a été décelé par contrôles de la mortalité, du gain de poids ponderal et des oocystes éliminées. Il a révélé une activité intrinsèque dépassant dix fois celle des autres agents coccidiostatiques disponibles, et le spectre d’activité est plus large que ce de la plupart d’eux. Die fortlaufende Verabreichung von Futter, das 0,001 Prozent dieses Präparates enthält, verhütet Kücken gegen Infektion mit Eimeria acervulina, E. brunetti, E. maxima, E. mivati, E. necatrix und E. tenella, was durch niedrige Mortalität, gute Wachstumszunahme und niedrige Oozystausscheidung bewiesen wurde. Die Verbindung, die chemisch 7-benzyloxy-6-n-butyl-3-methoxycarbonyl quinol-4-one ist, zeigte eine zehnfache Aktivität im Vergleich mit anderen kokzidiostatischen Verbindungen und auch eine grössere therapeutische Breite als die meissten Verbindungen, die zur Verfügung stehen. El compuesto (7-benziloxy-6-n-butil-3-metoxycarbonil quinol-4-one) addicionato continuamente al alimento al nivel de 0,001 por ciento ha producido una excelente protección contra Eimeria acervulina, E. brunetti, E. maxima, E. mivati, E. necatrix y E. tenella en pollitos, como evidenciado por controles de mortandad, incremento del peso corporeo y eliminación de oocistos. El ha mostrado más de diez veces de la actividad intrinseca de otros compuestos coccidiostaticos disponsibles, y tiene un espectro de actividad más amplio de el de la mayoría de esos.

Renewal of cell populations.

Renewal of cell populations.