Yolk amprolium levels produced by giving chickens amprolium in feed or water.
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The addition of liquid amprolium to the drinking water on days when medicated (amprolium) ration was not fed in a restricted feeding (skip-a-day) program improved protection against a primary exposure to Eimeria acervulina and Eimeria tenella, yet still allowed for the development of protective immunity to subsequent challenge. With E. tenella, the best protection, as measured by reduction of lesion score, was provided by amprolium given in the drinking water on alternate days to feed medication when compared with the use of amprolium only in the feed or liquid amprolium at less frequent intervals (every second or third nonfeeding day). With Eimeria maxima, amprolium in the feed did not significantly lower lesion score compared with the score in unmedicated pullets; however, the further addition of amprolium to the drinking water did. When pullets were reared in floor pens previously seeded with coccidia, amprolium medication in the feed alone reduced the E. tenella-induced mortality rate from 28 to 8%. The addition of amprolium in the drinking water on nonfeeding days eliminated all deaths. Floor-reared pullets were caged after 3 wk and challenged 1 wk later with the same species of coccidial oocysts used to immunize on the floor. Coccidial lesion scores following challenge were eliminated or markedly lower than in pen-reared (unimmunized) pullets similarly challenged. This indicated that protective immunity developed despite the use of amprolium in the drinking water.
Amprolium may be used as a coccidiostat in rearing hens and is a therapeutical agent used in laying hens. As a result of cross contamination, low amprolium levels may occur in feed. Feed containing a concentration of amprolium ranging from 5 to 250 mg/kg was therefore supplied to groups of laying hens. The amprolium residues in the yolks during and after treatment were subsequently determined. These levels varied from 1.75 mg/kg in the group fed 250 mg/kg to 0.2 mg/kg in the group fed 5 mg/kg. Amprolium levels in the whites of eggs were much lower than those in the yolks. The residues in yolks decreased below detectable levels (less than 0.005 mg/kg) within approximately ten days after treatment. Rearing hens in a tiered wire floor system were given amprolium in their feed until the first egg was laid. Amprolium residues in yolks were detected for well over a fortnight after the onset of laying. The amprolium residues determined in yolk did not exceed US tolerance levels of 8 mg/kg.
Amprolium reduced the number of oocysts shed by Eimeria acervulina, E. maxima, E. necatrix, and a mixture of susceptible strains of E. tenella. Sporulation of oocysts from mediated chickens was reduced compared with that of oocysts from unmedicated chickens. Sporulation was reduced by levels of 0.0250% amprolium for E. acervulina and by levels of 0.0060% for E. maxima and the susceptible E. tenella. Not enough oocysts were recovered to measure sporulation of E. necatrix. Sporulation reduction was not affected by the method of administration of amprolium (feed or water), except with E. acervulina, for which fewer oocysts sporulated when 0.0120% amprolium was added in the drinking water than when 0.0125% amprolium was added to the feed. Conversely, amprolium medication had no effect on the sporulation of an amprolium-resistant E. tenella. When fed to unmedicated chickens, those oocysts from amprolium-medicated chickens that did sporulate were as infective as oocysts recovered from unmedicated chickens.
Polioencephalomalacia was induced in eight buffalo calves, 6-12 months old, by drenching amprolium (300 mg/kg body weight per day) for 29-55 days. Four buffalo calves of the same age group were drenched with tap water only and served as control. Blood samples were collected at different intervals during amprolium administration until the onset of clinical signs. Cerebrospinal fluid was also collected prior to amprolium administration and at the onset of clinical signs. A significant progressive decrease in erythrocyte transketolase (TK) activity and an increase in the percent of thiamine pyrophosphate (TPP) effect were observed in amprolium-fed calves during amprolium administration until the onset of clinical signs. There was a significant increase in blood lactate and blood pyruvate concentrations and a significant decrease in lactate/pyruvate ratio at the onset of clinical signs. Serum electrolyte (Na, Ca, P, Mg) concentrations showed no significant changes. However, the serum potassium concentration had decreased significantly at the onset of signs. The cerebrospinal fluid analyses revealed a significant increase in lactate and pyruvate concentrations and lactate/pyruvate ratio in amprolium-fed calves. The electrolytes (Na, K, Ca, P and Mg) of cerebrospinal fluid did not show any change. It is concluded that oral administration of amprolium (300 mg/kg body weight daily) for 4-6 weeks produces biochemical changes characteristic of polioencephalomalacia in buffalo calves.
The development of resistance by the Houghton strain of Eimeria tenella to the anticoccidial drugs amprolium, clopidol and methyl benzoquate has been studied. Resistance to amprolium and clopidol developed more readily in experiments where a large number of coccidia were exposed to the drug, either by increasing the number of oocysts in the inoculum or by increasing the number of birds in the group. When 45 birds were given 2.0 X 10(6) oocysts, resistance to amprolium and clopidol appeared after 6 and 7 passages respectively. In previous experiments, under similar conditions, resistance to robenidine developed after 6 passages, suggesting little difference between these three drugs. Resistance to amprolium and clopidol arose gradually as the concentration of drug was increased, but resistance to methyl benzoquate appeared in a single step from sensitivity to high-level resistance. Both amprolium and clopidol-resistant lines showed an 8-fold reduction in drug sensitivity. Attempts to measure the degree of resistance by calculation of the ED50 were unsuccessful.
This study investigated the hypothesis that the consumption of egg yolks might lead to thiamin inadequacy in infants because of the possible contamination of the egg yolks with amprolium. Earlier workers showed that the presence of amprolium in the diet inhibits the absorption of thiamin. Amprolium is added to some poultry feeds to control coccidiosis: it is readily incorporated in the egg yolk and egg yolk is one of the solid foods offered to infants at weaning. We found that under current commercial poultry feeding practices in WA it is extremely unlikely that any amprolium would be present in commercial eggs or poultry. Amprolium was undetectable in eggs purchased at several retail outlets. Thus there is no evidence that consumption of egg yolk contributes to thiamin inadequacy in infants.
Polioencephalomalacia (PEM) induced in sheep was compared with the disease found in naturally occurring cases. Blood biochemical indicators measured were pyruvate, lactate, glucose, erythrocyte transketolase (TK) and stimulation of TK by addition of thiamine pyrophosphate (TPP effect). Faeces and rumen contents were assayed for thiaminase activity. The effect of treating affected sheep with thiamine was also noted. It was found that amprolium treatment could induce thrombocytopenia, but once the sheep became accustomed to amprolium in the diet they seemed to be resistant to this effect. In sheep receiving amprolium significant weight losses preceded the onset of clinical signs. Further weight loss continued throughout the recovery period despite removal of amprolium from the diet and treatment with thiamine. Blood glucose was variable, and was elevated only when marked clinical signs were present. Pyruvate and lactate levels showed marked variation throughout the trial. TK values were depressed and TPP effects increased well before the onset of clinical signs, although some naturally occurring cases had normal levels. Faecal thiaminase activity was negligible in all the sheep on the amprolium trial but most field cases had a high level. High faecal thiaminase was observed in about 5% of clinically normal animals from affected flocks. Depression of erythrocyte TK activity coupled with the presence of faecal thiaminase appeared to be the most reliable diagnostic biochemical parameters for PEM. Treatment of PEM affected sheep with thiamine rapidly brought the biochemical status of the animals to normal. However where advanced brain lesions were present the damage was permanent and such sheep treated with thiamine remained partially decorticate.
A continuous flow system was coupled to a high-pressure liquid chromatography (HPLC) system, resulting in an automated system for the determination of amprolium in egg yolk and (chicken) muscle tissue. The sample was diluted (yolk) or extracted (tissue) with water, and the solution obtained was dialysed against water as the recipient stream. Aliquots of the dialysed solutions were pumped onto a short pre-concentration column. By means of the mobile phase, the concentrate was back-flushed onto the analytical column and amprolium was separated from interfering substances, using a reversed phase ion-pair system. Amprolium was post-column oxidized to amprochrome, which was detected fluorometrically. Linear calibration curves for both yolk an muscle tissue were obtained in the 10-250 micrograms/kg range. The detection limit is approximately 3 micrograms/kg. This method was applied to eggs and muscle tissue, which were commercial obtained. Egg yolk was found to be frequently contaminated with low levels of amprolium (29.4% positive of 266 samples investigated; mean concentration of positive samples = 58 micrograms/kg), whereas only a few muscle samples contained detectable levels (4.9% positive of 81 samples investigated; mean concentration of positive samples = 5 micrograms/kg).
Two extraction and liquid chromatographic procedures are presented which separate amprolium from compounds in poultry feed or premixes that could interfere with its fluorometric determination. The procedures are based on earlier work on the determination of thiamine in food samples. Amprolium is extracted from feed with a hexane-aqueous sulfosalicylic acid mix, separated on a C18 column, and detected fluorometrically after postcolumn derivatization. For premixes, water extraction is used. Values for the amprolium content of poultry feed obtained with these procedures are in good agreement with those obtained with AOAC official methods. It is suggested that these methods with suitable modifications may be of use for routine analysis of amprolium in feeds. The overall methods are rapid and appear to give reasonable results.
We have observed that treatment with high concentrations of a thiamine analog (amprolium) can lead to the elimination of the plasmidic resistance to ampicillin and the production of enterotoxin in wild Escherichia coli strains and in E coli and Salmonella typhimurium strains which had received the pKM101 plasmid through bacterial conjugation. By computer analysis, we also have determined that there is a highly significant (P less than 0.01) synergism between ampicillin and amprolium which reduces considerably the growth of certain enteric bacterial strains which have a plasmidic resistance to ampicillin and which were not markedly affected by amprolium alone, in our experimental conditions. Our data indicate that the rate of loss of the plasmid pKM101 after treatment of R+ bacterial strains with amprolium can be increased.
Amprolium administered in feed during the first 4 weeks of life at a level of 0.0175% protected pheasants against three major pathogenic species of coccidia (Eimeria colchici, E. duodenalis, and E. phasiani) when they were exposed at 2 weeks of age. The difference was significant when mortalities were compared between medicated infected (3%) and unmedicated infected (35%) pheasants. The manufacturer's proposed level (0.0175%) and twice the proposed level (0.0350%) of amprolium had no significant effect on weight gains or mortality in the safety trial. Amprolium residues found in the muscles and livers of pheasants that received either level of amprolium did not exceed the tolerance levels for chickens and turkeys permitted by the U.S. Food and Drug Administration.
An experiment was conducted to investigate the mechanisms responsible for the brain lesions, diarrhoea and haemorrhages produced by amprolium poisoning of preruminant lambs. The encephalopathy was preceded by a reduction of cerebrocortical transketolase activity. Diarrhoea was not associated with histological evidence of pathological change in the small intestine, and in lambs with severe diarrhoea the small intestinal levels of alkaline phosphatase and lactase were unaffected. Haemorrhages were associated with a thrombocytopenia which was attributed to degeneration of bone marrow megakaryocytes. The bone marrow of the majority of the lambs which had received amprolium for 3 wk or more was severely depopulated, erythrocyte precursors being the most severely affected. The marrow depopulation was atrributed to decreased cell production, as the majority of the remaining cells showed little evidence of degeneration and the number of mitotic figures in the marrow of amprolium-treated lambs was considerably reduced as compared with the controls.
Sulphadimidine, amprolium, halofuginone and chloroquine phosphate were administered to buffalo calves 10 days after experimental infection with Eimeria bareillyi. Animals given sulphadimidine or amprolium remained clinically normal and shed only a few oocysts in their faeces. Halofuginone was found partially effective and chloroquine phosphate completely ineffective in preventing faecal oocyst discharge and intestinal lesions. Sulphadimidine and amprolium treated calves gained weight, but chloroquine treated calves suffered progressive weight loss similar to that of infected untreated controls. No significant alterations of haematological values were observed either in the treated calves or in the untreated controls.
Amprolium and carbarsone were tested separately and in combination for efficacy in prevention of occidiosis and histomoniasis, respectively. In these studies, amprolium at .0125% in the diet was efficacious in prevention of coccidiosis, and carbarsone had no deleterious effects when fed simultaneously at .0375%. Carbarsone at .0250% was efficacious in prevention of histomoniasis, and amprolium had no deleterious effects when fed concurrently at .025%. These results indicated that both drugs could be used in combination without interference of efficacy against the target diseases.
Amprolium [1-(4-amino-2-propyl-5-pyrimidinemethyl)-2-methyl-pyridinium chloride hydrochloridel is a basic (quaternary) organic compound. At very low plasma concentration, it is cleared by the kidney at a rate approximating renal plasma flow in the dog. Its renal clearance is not depressed by organic acids (p-aminohippurate or probenecid) but is reduced by the quaternary base, mepiperphenidol. Acetate and pantothenate may influence the clearance of amprolium but, if this is the case, the effect is less than for p-aminohippurate. Its clearance is depressed as urinary pH is increased. The clearance of amprolium was not altered over a substantial range in urine flow at either high or low urinary pH.
The efficacy of preventive in-feed medication with amprolium (2000 ppm) was studied on a farm where clinical coccidiosis in unweaned lambs at pasture has been a problem for the past seven years. Both treated and untreated control lambs had access to the concentrates through creep feeding. In this clinical trial neither the treated group (15-17 mg of amprolium per kg body weight per day for three weeks) nor the control group showed clinical symptoms of coccidiosis. It seems likely that this is attributable to the feeding of concentrates. Nevertheless, the excretion of oocysts by the animals of the treated group was significantly lower than that of the control group. An outbreak of clinical coccidiosis in another group of lambs on this farm was successfully controlled by single drenching, 50 mg.kg-1, followed by the medicated feed. The pharmaceutical availability of amprolium in the concentrates was 95 +/- 1% immediately after preparation and the stability during storage under field conditions for two months was 100% +/- 2%.
A method is presented for determination of amprolium residues in chicken muscles by a liquid chromatographic post-column reaction system. The drug is extracted from muscles with methanol, and the extract is concentrated to 3-4 mL. This aqueous solution is rinsed with n-hexane and cleaned up by alumina column chromatography. The drug is separated from the interferences on a LiChrosorb RP-8 column, reacted with ferricyanide in alkaline solution, and quantitated by fluorometric detection at 367 nm (excitation) and 470 nm (emission). Recoveries of amprolium added to chicken muscles at levels of 0.1 and 0.2 ppm were 74.9 and 80.9%, respectively. The detection limit was 1 ng for amprolium standard and 0.01 ppm in chicken muscles.