Eimeria tenella: anticoccidial action of drugs in birds with surgically closed ceca.

Surgical ligation of chick ceca was used to study the role of absorption and extraintestinal transport in the action of anticoccidial drugs. The administration of drugs in the feed was started after ligation of one of the paried ceca. Birds were inoculated orally with oocysts of Eimeria tenella before cecal ligation or were given bilateral cecal injections of sporozoites after ligation. Cecal lesions caused by the coccidia were evaluated and compared on day 6 postinoculation. Lesions in ligated and unligated ceca were reduced by feeding robenidine (33 ppm), arprinocid (70 ppm), zoalene (125 ppm), aklomide (250 ppm), clopidol (125 ppm), nicarbazin (125 ppm), monensin (120 ppm), salinomycin (60 ppm), and lasalocid (75 ppm). The lesions were more severe in the ligated cecum than in the intact cecum, whether in nonmedicated or medicated birds, but the differences were statistically significant only upon treatment with amprolium, aklomide, robenidine, and clopidol. Generally, however, all drugs except amprolium, significantly reduced the lesions in the ligated cecum in comparison with the control, nonmedicated ligated cecum. Therefore, we concluded that the systemic absorption of most anticoccidial drugs contributes significantly to their efficacy against coccidia in the intestinal mucosa.     

Eimeria tenella: experimental studies on the development of resistance to amprolium, clopidol and methyl benzoquate.

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.     

Factors influencing the assessment of anticoccidial activity in cell culture

A comparative study has been made of the factors influencing the assessment of anticoccidial potency in vitro against Eimeria tenella using established anticoccidials and arprinocid and some of its analogues. Drugs whose potency depended upon medium composition were amprolium, lasalocid and halofuginone. There was a difference in strain sensitivity with robenidine. Host cell type had an important effect on potency of monensin, decoquinate, arprinocid and its analogues. Arprinocid was active in chick liver cell systems but totally inactive in chick kidney cell systems, although its N-oxide was active in both cell types. Arprinocid-containing medium, conditioned by supporting the growth of chick embryo liver cell cultures, had an anticoccidial effect on E. tenella growing in chick kidney cells. It is deduced that the anticoccidial activity of arprinocid in the chick is due to a metabolite.     

Eimeria tenella: immunogenicity of arrested sporozoites in chickens

Groups of chickens were medicated with the anticoccidial drug, decoquinate, and starting 1 day after this medication they were given daily inoculations of either 1 X 10(4) (Experiment 1) or 1 X 10(5) (Experiment 2) oocysts of a decoquinate-sensitive strain of Eimeria tenella. This assured the presence of large numbers of drug-inhibited sporozoites in the cecal tissues. The immunity arising from the presence of these inhibited sporozoites was assessed by challenging the medicated chickens with a 2.5 X 10(5) oocysts of a decoquinate-resistant strain of E. tenella. The response to challenge was assessed by weight gain, the severity of cecal lesions, hematocrits, and cecal oocyst numbers. The inhibited sporozoites promoted little (if any) immunity judged by clinical signs of disease. However, judged by body weight changes after challenge, the presence of inhibited sporozoites provided substantial protection against the body-weight-depressing effects of the challenge dose. These findings emphasize the importance of stage-specific antigen expression in Eimeria spp. infections and support the notion that immunogenicity is associated with tropic stages of the parasite.     

Effect of dietary zinc level on serum carotenoid levels, body and shank pigmentation of chickens after experimental infection with coccidia

Two experiments were conducted to test the effects of a dietary zinc amino acid complex (Zn-AA) and an anticoccidial drug on Eimeria acervulina or Eimeria tenella infections. In each experiment, 288 day-old Three-Yellow-Chickens were used in a 2 x 3 factorial experimental design. Six groups were arranged randomly to receive three levels of Zn-AA (0, 40, or 80 mg/kg) alone or with salinomycin (60 mg/kg). Additionally an uninfected group was set as negative control. At the age of 21 days birds in Exp. 1 were inoculated with 3 x 10(4) sporulated E. acervulina oocysts, while birds in Exp. 2 were inoculated with 1.5 x 10(4) sporulated E. tenella oocysts. In Exp. 1, E. acervulina did not suppress growth performance significantly, but in groups without salinomycin it significantly reduced serum carotenoid levels on day 7 after inoculation and body and shank pigmentation on day 42. Salinomycin medication maintained serum carotenoids and visual colour of inoculated birds, but Zn-AA did not influence these parameters. In Exp. 2, growth performances of infected and uninfected chickens were similar. Infection decreased to only serum carotenoid levels on day 14 after infection, and colour scores on day 42 in the inoculated group without salinomycin and Zn-AA supplementation. The birds that received Zn-AA had significantly higher serum carotenoid levels and colour scores than those that did not. Although supplementation of Zn-AA cannot avoid coccidial damage of caecum, it prevents the reduction of serum carotenoids and pigmentation of Three-Yellow-Chicken infected with E. tenella, but not after infection with E. avervulina. The interactive effects between Zn-AA and salinomycin on growth performance and pigmentation were not significant.     

Effect of flubendazole on the number of first-stage larvae of Angiostrongylus cantonensis released in the faeces of treated rats

The effect of flubendazole orally administered at 10 mg/kg/day for 5 consecutive days (the 11th, 20th or 40th post-infection) on the number of first-stage larvae (L1) of Angiostrongylus cantonensis released in the faeces of rats each infected with 40 third-stage larvae was determined. Faecal examination for 5 months, the period from medication to dissection of rats, showed that L1 release ceased in all the rats of medicated groups by about 1 week after the termination of dosing and resumed 1-2 months later in 86% of the rats which were dissected at the end of experiments with the recovery of adult worms of both sexes. Throughout the period of 5 months, about 2-4 x 10(4) L1/gram of fresh faeces was recorded in non-medicated control groups. There was a 38-79% reduction in adult worms at the dissection. Microscopic examination of the uteri of the remaining adult worms and lung tissues of rats confirmed no normal egg production in the adult worms from rats of medicated groups, except the rats with the resumption of faecal L1 release.     

Eimeria tenella, E. necatrix, E. acervulina, E. maxima, and E. brunetti: potent anticoccidial activity of an uridine analog, 1-(beta-D-ribofuranosyl)-2(1H)-pyrazinone 4-oxide

The anticoccidial activity of an uridine analog, 1-(beta-D-ribofuranosyl)-2(1H)-pyrazinone 4-oxide (emimycin riboside), against five species of chicken Eimeria was tested individually in battery experiments. With 16 ppm of the compound in feed, marked anticoccidial activity was obtained against Eimeria tenella, E. necatrix, E. acervulina, E. maxima, and E. brunetti. The last named species was more drug-sensitive than the others--dietary levels of at least 8 ppm of the drug exhibited good protection and eliminated practically all clinical signs. The battery tests with delayed and restricted medications showed that emimycin riboside affected the development of parasites in first and second generation schizogony of the life cycle of E. tenella.     

In vivo anticoccidial,antioxidant, and anti-inflammatory activities of avocado fruit,Persea americana (Lauraceae), against Eimeria papillata infection

Apicomplexan parasites, especially Eimeria sp., are the main intestinal murine pathogens, that lead to severe injuries to farm and domestic animals. Many anticoccidial drugs are available for coccidiosis, which, leads to the development of drug-resistant parasites. Recently, natural products are considered as an alternative agent to control coccidiosis. This study was designed to evaluate the anticoccidial activity of the Persea americana fruit extract (PAFE) in male C57BL/6 mice. A total of 35 male mice were divided into seven equal groups (1, 2, 3, 4, 5, 6, and 7). At day 0, all groups except the first group which served as uninfected-untreated control were infected orally with 1 × 10³ E. papillata sporulated oocysts. Group 2 served as uninfected-treated control. Group 3 was considered an infected-untreated group. After 60 min of infection, groups 4, 5, and 6 were treated with oral doses of PAFE aqueous methanolic extract (100, 300, and 500 mg/kg of body weight, respectively). Group 7 was treated with amprolium (a reference drug for coccidiosis). PAFE with 500 mg/kg, was the most effective dose, inducing a significant reduction in the output of oocysts in mice feces (by about 85.41%), accompanied by a significant decrease in the number of the developmental parasite stages and a significant elevation of the goblet cells in the jejunal tissues. Upon treatment, a significant change in the oxidative status due to E. papillata infection was observed, where the levels of glutathione (GSH) increased, while, levels of malondialdehyde (MDA) and nitric oxide (NO) were decreased. In addition, the infection significantly upregulated the inflammatory cytokines of interleukin-1β (IL-1β), tumor necrosis factor-alpha (TNF-α), and interferon-γ (IFN-γ). This increase in mRNA expression of IL-1β, TNF-α, and IFN-γ was about 8.3, 10.6, and 4.5-fold, respectively, which significantly downregulated upon treatment. Collectively, P. americana is a promising medicinal plant with anticoccidial, antioxidant, and anti-inflammatory activities and could be used for the treatment of coccidiosis.     

Digestive and metabolic utilization of lauric,myristic and stearic acid in cows,and associated effects on milk fat quality

A study was carried out to examine the effect of dietary supplementation of oregano essential oil on performance of broiler chickens experimentally infected with Eimeria tenella at 14 days of age. A total of 120 day-old Cobb-500 chicks separated into 4 equal groups with three replicates each, were used in this study. Two groups, one infected with 5·10⁴ sporulated oocysts of E. tenella and the other not, were given a basal diet and served as controls. The other two groups also infected with E. tenella were administered diets supplemented with oregano essential oil at a level of 300 mg/kg, or with the anticoccidial lasalocid at 75 mg/kg. Following this infection, survival rate, bloody diarrhoea and oocysts excretion as well as lesion score were determined. Throughout the experimental period of 42 days, body weight gain and feed intake were recorded weekly, and feed conversion ratios were calculated. Two weeks after the infection with E. tenella supplementation with dietary oregano oil resulted in body weight gains and feed conversion ratios not differing from the non-infected group, but higher than those of the infected control group and lower than those of the lasalocid group. These parameters correspond with the extent of bloody diarrhoea, survival rate, lesion score and oocyst numbers and indicated that oregano essential oil exerted an anticoccidial effect against E. tenella, which was, however, lower than that exhibited by lasalocid.     

Synthesis and SAR studies of diarylpyrrole anticoccidial agents

2-(4-Fluorophenyl)-3-(4-pyridinyl)-5-substituted pyrroles were prepared and evaluated as anticoccidial agents in both in vitro and in vivo assays. Among the compounds evaluated, the dimethylamine-substituted pyrrole 19a is the most potent inhibitor of Eimeria tenella PKG (cGMP-dependent protein kinase). Further SAR studies on the side chain of the 2-pyrrolidine nitrogen did not enhance in vivo anticoccidial activity.     

Studies on short-term application of drugs to Eimeria tenella in tissue culture.

The anticoccidial action of 6 drugs was studied by treating cell cultures with drug for 24 h after inoculation of the cultures with sporozoites of Eimeria tenella. A monitoring technique was used to confirm that the drug was being removed. Only monensin was shown to be coccidiocidal. Amprolium, lasalocid, methyl benzoquate, nicarbazin and robenidine were coccidiostatic at their minimum effective doses. Misleading coccidiocidal effects were observed when higher concentrations of methyl benzoquate, nicarbazin or robenidene were used.     

Eimeria tenella,E. necatrix,E. acervulina,and E. maxima: Anticoccidial activity of 1,6-dihydro-6-oxo-2-pyrazinecarboxylic acid 4-oxide

The anticoccidial activity of an orotic acid analog, 1,6-dihydro-6-oxo-2-pyrazinecarboxylic acid 4-oxide (carboxyemimycin), was tested in battery experiments, utilizing 9-day-old Single-Comb White Leghorn cockerels. Carboxyemimycin, at 125 ppm and more in feed, exhibited marked anticoccidial activities against Eimeria tenella, E. necatrix, E. acervulina, and E. maxima. High doses of carboxyemimycin—up to 1000 ppm—did not cause any reduction in weight gains. The battery and in vitro studies with delayed and restricted medications revealed that carboxyemimycin affected the development of E. tenella in first and second generation schizogony and in gametogony.     

Expression of flotillin-1 on Eimeria tenella sporozoites and its role in host cell invasion

Lipid rafts are detergent-resistant, liquid-ordered microdomains in plasma membranes that are enriched in cholesterol and sphingolipids and involved in intracellular signal transduction, membrane trafficking, and molecular sorting. In this study, we investigated the possibility that lipid rafts on Eimeria tenella sporozoites may act as platforms for host cell invasion. Flotillin-1, a resident protein of lipid rafts, was identified on E. tenella sporozoites and was prominently expressed at the apex of the cells, a region mediating host cell invasion. Pretreatment of sporozoites with antibody against flotillin-1 blocked parasite invasion. Furthermore, the anticoccidial drug, monensin, disrupted the localization of flotillin-1 within raft structures resulting in loss of invasion. We conclude that Eimeria sporozoites utilize lipid rafts containing flotillin-1 for internalization into host cells.     

Comparison Between a Live,Attenuated Anticoccidial Vaccine and an Anticoccidial Ionophore,on Performance of Broilers Raised with or without a Growth Promoter,in an Initially Eimeria-Free Environment

An experiment was carried out to study the effects of vaccination with Paracox®, a live, attenuated vaccine against avian coccidiosis, on broilers isolated from extraneous Eimeria parasites. The study involved 3200 broiler chickens raised in floor pens similar to commercial conditions, but in an initially Eimeria-free environment. Forty percent of the chickens were vaccinated at 3 days of age and given either a basal unmedicated feed or a feed supplemented with the feed antibiotic virginiamycin. Unvaccinated birds were given either the basal feed or feed supplemented either with virginiamycin or the anticoccidial ionophore narasin. At slaughter at 36 days of age vaccinated birds had a lower live weight than non-vaccinated birds. The difference was 4.6% in unmedicated, and 6.0% in virginiamycin medicated chickens. Feed conversion ratio at slaughter was 2.5% higher for unmedicated vaccinated birds, and 1.3% higher for virginiamycin medicated vaccinated birds, compared to respective non-vaccinated groups. There was no significant difference in overall performance of unvaccinated birds given narasin as compared to virginiamycin. At 10 days post vaccination vaccinated birds had a higher number of Clostridium perfringens in the caeca, but there was no difference thereafter. Throughout the experiment, caecal clostridial counts were considerably higher in vaccinated unmedicated birds than in unvaccinated birds given narasin. The number of oocysts shed in the vaccinated groups was very low, but during a subsequent challenge with E. maxima and E. tenella the birds’ immunity was found to be satisfactory.     

E.tenella感染雏鸡特异性抗体的动态变化和母源免疫的研究

本实验用斑点酶联免疫试验 (Dot ELISA)检测了E .tenella感染雏鸡特异性抗体、免疫母鸡卵黄和后代血清中特异性母源抗体的动态变化及不同日龄雏鸡的抗球虫水平。结果表明 :(1)雏鸡感染E .tenella后第 6d即可在循环血液中检测到特异性IgG ,于第 18天达到峰值 ,第 30天降至感染后第 7天时的水平。 (2 )母鸡二次免疫后第 5天卵黄中即有高水平的母源抗体 ,免疫母鸡后代 1日龄时母源抗体滴度高达 9.83,与未免疫对照组差异极显著 (P <0 .0 1) ,二者都随着日龄增长降低 ,但免疫组下降幅度很小 ,2 9日龄时仍和对照组 18日龄水平相当。 (3)免疫母鸡后代对再感染的抵抗力显著增强 ,卵囊减少率高达 93%,抗球虫指数和相对增重率都与对照组差异极显著 (P <0 .0 1) ,雏鸡的抗球虫水平随着日龄增长逐渐降低 ;母源抗体、抗球虫指数和相对增重率三者之间两两呈现明显的正相关 ,而这三者又都与卵囊产量呈明显的负相关。     

Evaluation of herbal coccidiostat 'Coxynil' in broiler

Anticoccidial efficacy of "Coxynil" a polyherbal preparation was tested against Eimeria tenella in broilers. Body weight of birds challenged with E. tenella in Coxynil treated groups was higher as compared to Coxynil untreated. Oocyst out put, lesion score, HI titres against New Castle disease virus were significantly higher in Coxynil supplemented groups in comparison to Coxynil un-supplemented groups. Examination of ceaca of the birds, revealed that the Coxynil interfered with life cycle of coccidia. The typical second generation schizonts were absent in ceacal section of Coxynil treated groups. The results indicate that Coxynil is effective herbal coccidiostat.     

Dihydrofolate Reductase from Eimeria tenella: Rationalization of Chemotherapeutic Efficacy of Pyrimethamine

SYNOPSIS. Dihydrofolate reductase activity of 0.2 nmole of dihydrofolate reduced/min/mg protein was detected in crude extracts of unsporulated oocysts of Eimeria tenella. The enzyme was purified by a combination of affinity and ion-exchange chromatography. Its molecular weight was estimated as 240,000 daltons. An anticoccidial drug pyrimethamine is a potent inhibitor of the activity of E. tenella dihydrofolate reductase ( K _t = 3 nM), but it is less effective an inhibitor of dihydrofolate reductase from chicken liver. This difference may explain the in vivo therapeutic action of pyrimethamine against Coccidia.     

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.     

Synthesis and SAR of 2,3-diarylpyrrole inhibitors of parasite cGMP-dependent protein kinase as novel anticoccidial agents

Several analogs of 2,3-diaryl pyrroles were synthesized and evaluated as inhibitors of Eimeria tenella cGMP-dependent protein kinase and in in vivo anticoccidial assays. A 4-fluorophenyl group enhances both in vitro and in vivo activities. The most potent analogs are the 5-(N-methyl, N-ethyl, and N-methylazetidine methyl) piperidyl derivatives 12, 23, and 34. These compounds have a broad spectrum of activity. Based on the in vivo efficacy and cost of synthesis, the N-ethyl analog 23 was chosen as a novel anticoccidial agent for a field trial.     

Eimeria tenella: effects of immunity on sporozoites within the lumen of the small intestine

The effect of immunity on the numbers of sporozoites of Eimeria tenella recoverable from the lumen of the small intestine 1 hr after an oral challenge inoculum of oocysts was examined. The experiments were carried out in chickens which had been given an immunizing inoculum of oocysts 9 or 18 days earlier, and the results were compared with those obtained in a control, unprimed, but similarly challenged, group. Similar numbers of "challenge" sporozoites were found in the intestinal washes of control and 18 day primed chickens but there were fewer in the 9 day primed groups. The titers of antisporozoite IgA antibodies (measured by indirect fluorescence) were higher in the gall bladder bile of the 9 day primed groups but resistance to reinfection (measured by the output of oocysts in the feces after challenge with oocysts orally or with sporozoites intracecally) was greater in the 18 day primed group. Although fewer in number, the challenge sporozoites recovered from the intestinal washes of 9 day primed chickens appeared to be morphologically normal when examined by light microscopy. Also, they were as infective as sporozoites recovered from unprimed control, or 18 day primed, groups when injected intracecally into naive chickens. The findings indicate that, whereas reduction of the number of sporozoites of E. tenella in the lumen of the small intestine (presumably caused by the action of secreted antibodies) can be a means of reducing the effective challenge inoculum, this mechanism does not play a major role in the expression of immunity.