Anticoccidial activity of 8-aminoquinolines, pamaquine, primaquine and several molecular complexes and salts of pamaquine, against Eimeria tenella, E. necatrix, E. acervulina, E. maxima, and E. brunetti in battery experiments.

Pamaquine and primaquine, the well known antimalarial 8-aminoquinolines, have not been reported for their anticoccidial activity. A series of battery experiments was conducted to investigate their activity against a laboratory strain of Eimeria tenella, E. necatrix, E. acervulina, E. maxima, or E. brunetti and revealed that both drugs were effective against E. tenella and E. necatrix, but not against the other three species. Pamaquine suppressed the symptoms of E. tenella induced coccidiosis at concentrations above 125 ppm in feed and primaquine controlled the clinical signs as well at levels above 31.2 ppm. The activity against E. necatrix was observed with pamaquine at 250 ppm and with primaquine at levels above 125 ppm. Pamaquine showed a tendency apparently to reduce body weight gain at 125-500 ppm, whereas primaquine showed the same tendency at 500 ppm. In a concomitantly conducted experiment, this adverse effect of pamaquine was averted in its molecular complexes with benzophenone, nitropyrazole, dinitrobenzoic acids and quinoline, and in its salts of sulfate or zinc chloride, and yet these compounds retained the same anticoccidial activity as of pamaquine. This suggests that these compounds had the broadened safety margin. Judging from their susceptibility to these compounds. E. tenella and E. necatrix will have similar metabolic functions to those of blood cell parasitizing protozoa like plasmodia and prioplasma, which are easily suppressed by this class of compound.     

Isolation and selection of ionophore-tolerant Eimeria precocious lines: E. tenella, E. maxima and E. acervulina

Eimeria parasites were isolated from Nanhai Guangdong province (southern China) and studied in chickens in wire cages to evaluate their drug resistance against commonly used ionophores: monensin (100 mg/kg of feed), lasolacid (90 mg/kg), salinomycin (60 mg/kg), maduramicin (5 mg/kg) and semduramicin (25 mg/kg). Chinese Yellow Broiler Chickens were infected with 40,000 crude sporulated Eimeria oocysts at 15 days of age and prophylactic medication commenced a day prior to infection. Drug resistance was assessed for each ionophore drug by calculating the anticoccidial index (ACI) and percentage optimum anticoccidial activity (POAA) based on relative weight gain, rate of oocyst production and lesion values. Results revealed that Nanhai Eimeria oocysts comprising of E. tenella, E. maxima and E. acervulina, were resistant to monensin, sensitive to both salinomycin and lasolacid and partially sensitive to maduramicin and semduramicin. By selection for early development of oocysts during passage through chickens, the prepatent time of E. tenella, E. maxima and E. acervulina were reduced by 49, 36 and 22 h, respectively. The precocious lines are less pathogenic than the parent strains from which they were selected and conferred a satisfactory protection for chickens against coccidiosis. These ionophore-tolerant precocious lines could have wider applications in the development of anticoccidial vaccines for sustainable control of coccidiosis.     

鸡胚接种柔嫩艾美耳球虫和堆形艾美耳球虫活卵囊的免疫保护试验

给18日龄鸡胚接种一定剂量的柔嫩艾美耳球虫(Eim eria tenella)和/或堆形艾美耳球虫(E.acervulina)孢子化卵囊,出雏后在无球虫环境中笼养,1~10日龄每天收集各组粪便样本,计数克粪便卵囊数(OPG),并于14日龄时以大剂量同源孢子化卵囊攻虫,以相对增重率(RWG)、饲料转化率(FCR)、相对卵囊产量(ROP)评价免疫保护效果。结果显示,以E.tenella或E.acervulina卵囊免疫18日龄鸡胚,其卵囊排出的潜隐期及达到峰值的时间与1日龄雏鸡接种组相一致,有相似的排卵囊曲线,提示其诱导免疫的建立是在出雏后开始建立的。攻虫后各免疫组的RWG由攻虫对照组的31.9%~51.7%提高到了76.5%~83.6%,RCR由攻虫对照组的4.11~4.89改善为2.72~2.96,ROP降至4.7%~23.5%。结果表明以一定剂量E.tenella和E.acervulina卵囊单独或混合经羊膜腔免疫18日龄鸡胚都可以建立起针对出雏后14日龄同源攻虫的良好免疫保护力。比较混合免疫E.tenella和E.acervulina卵囊组与单一接种E.tenella或E.acervulina卵囊组的免疫效果发现,混合免疫组的各项指标均稍优于后者。     

Comparison of disease susceptibility and subclass-specific antibody response in SC and FP chickens experimentally inoculated with Eimeria tenella, E. acervulina, or E. maxima

Susceptibility to disease and the subclass-specific antibody response to Eimeria tenella, E. acervulina, and E. maxima were compared in two inbred strains of chickens, FP (B15B21) and SC (B2B2). FP strain was more susceptible to coccidiosis than SC chickens based on oocyst production, lesion score, and clinical signs. FP chickens infected with E. tenella had more severe cecal lesions and a significantly lower hematocrit level than SC chickens. FP chickens infected with E. acervulina excreted five times as many oocysts at 6 days postinfection as SC and showed a 71% reduction in plasma carotenoid level compared with controls (56% reduction in SC chickens). Body-weight change did not correlate with other signs of disease. Both SC and FP chickens produced high levels of serum IgM and IgG and biliary IgA. Although SC chickens had a slightly higher antibody response than FP chickens at 7 days postinoculation, both strains maintained high levels of IgM, IgG, and IgA for a prolonged period post primary inoculation. Although SC and FP chickens show different disease susceptibility to coccidiosis, they demonstrate similar antibody response.     

多重PCR检测3种鸡球虫方法的建立

根据GenBank中发表的巨型艾美耳球虫、柔嫩艾美耳球虫和堆型艾美耳球虫TS-1序列,设计了3对引物,建立了这3种球虫的单一PCR和多重PCR检测方法,并分别对单一PCR和多重PCR方法的特异性和敏感性进行了研究,对巨型艾美耳球虫、柔嫩艾美耳球虫和堆型艾美耳球虫的混合卵囊进行了初步应用.结果显示:单一PCR和多重PCR均能扩增出巨型艾美耳球虫、柔嫩艾美耳球虫和堆型艾美耳球虫特异性条带,其大小分别为151 bp、463 bp、303 bp,其最小检测浓度为0.5ng,对水牛梭形肉孢子虫、有毒艾美耳球虫、猪源弓形虫汤山株及其田间分离株均不起反应,表明建立的方法具有很强的特异性和较高的敏感性,可望用于巨型艾美耳球虫、柔嫩艾美耳球虫和堆型艾美耳球虫的诊断和田间种类调查.     

鸡柔嫩艾美耳球虫和巨型艾美耳球虫双重PCR检测方法的建立

为了建立一种能够快速实现对鸡柔嫩艾美耳球虫(Eimeria tenella)和巨型艾美耳球虫(E.maxima)同时进行检测的双重PCR方法,基于2种艾美耳球虫各自的ITS-1基因筛选2对特异性引物,对制备的阳性DNA样品进行PCR扩增,凝胶电泳检测结果显示,分别在约450bp和150bp处出现目的条带。对PCR反应中的退火温度、MgCl2浓度、DNA模板量等进行优化。结果表明,当退火温度为59℃、MgCl2浓度为2.5mmol/L、DNA模板量为2μL时,双重PCR扩增结果最佳。特异性检测结果显示,所建立的双重PCR对环形泰勒虫、羊巴贝斯虫、隐孢子虫和蓝氏贾第虫的扩增结果均呈阴性,表明建立的方法具有较高的特异性。成功建立了柔嫩艾美耳球虫和巨型艾美耳球虫双重PCR检测方法,为临床中鸡球虫病的快速诊断提供了一种可靠的检测方法。     

The extraintestinal stages of Eimeria tenella and E. maxima in the chicken

Donor chickens given feed medicated with one or two levels of decoquinate or given non-medicated feed were infected with oocysts of Eimeria tenella or E. maxima per os. Twelve hours after inoculation with oocysts liver, mid-intestine or ceca homogenates were fed to previously uninfected recipient chickens. The results showed that continuous medication with decoquinate was effective in preventing the transfer of sporozoites from the intestine to the liver. Oocysts were detected in the feces of all recipients of tissue from non-medicated donors, showing that some sporozoites of E. maxima and E. tenella are normally transferred to liver. Young broiler chickens were immunized by oral inoculation of E. maxima oocysts. The immune status of similar chickens inoculated with sporozoites of the same species directly into the liver or spleen were assessed. During the experimental period half of the chicks were provided with non-medicated food and the remainder were given feed supplemented with decoquinate; decoquinate was effective in arresting the development of the sporozoites. Two weeks after initial infection the birds were challenged with oocysts of E. maxima per os. Injection of sporozoites into the spleen did not protect against challenge. Birds inoculated with sporozoites into the liver were unable to develop a significant level of immunity. When the drug pressure was removed from these birds, parasitism of the intestine occurred and immunity developed.     

Effects of Natustat supplementation on performance, feed efficiency and intestinal lesion scores in broiler chickens challenged with Eimeria acervulina, Eimeria maxima and Eimeria tenella

The effects of dietary supplementation of Natustat™, a propriety plant derived product (Alltech Inc., KY, USA) and Salinomycin, on performance, feed efficiency and intestinal lesion scores were observed during two Eimeria challenge trials in broiler chickens. In the first trial chickens were challenged with Eimeria sp. via infecting the litter with a known amount of Eimeria oocysts. In the second trial the source of the Eimeria challenge was the litter from the first trial and the same treatment groups were assigned to the same pens as in the initial trial.

Birds were placed 55 per pen with seven pens per treatment. Performance parameters were recorded on days 21 and 42 during both trials. Intestinal lesion scores were assessed on days 14 and 21 during Trial 1 and on day 21 during Trial 2. Average weight gain and feed conversion ratios were significantly improved in the Natustat™ and Salinomycin treatment groups when compared to the non-supplemented infected group. Furthermore, lesion scores were lower on all sampling days in the Natustat™ and Salinomycin groups when compared to the non-supplemented group. However, only lesions associated with Eimeria tenella were significantly lowered by Natustat™ and Salinomycin supplementation.

Natustat™ and Salinomycin were equivalent in alleviating the negative performance effects associated with coccidiosis challenge. In summary, Natustat™ has the potential to be used as a natural alternative to chemotherapeutic drugs for Eimeria control.     

Studies on the stage of action of lasalocid against Eimeria tenella and Eimeria acervulina in the chicken

Broiler chickens in battery pens were either fed a diet containing 100 ppm lasalocid or no drug for 24 h prior to inoculation with sporulated oocysts of Eimeria tenella or Eimeria acervulina. Different groups of birds remained on medicated feed for 24, 48, 72, 96, 120 or 144 h after inoculation. Conversely, other groups started on an unmedicated diet, were given medicated feed at different times after oocyst inoculation. Starting lasalocid medication 24 h (E. tenella) or 48 h (E. acervulina) after inoculation reduced the lesions and improved the weight gain. There was no significant difference in performance of birds after withdrawal of the drug at 48 h (E. tenella) or 72 h (E. acervulina) and thereafter. Starting lasalocid medication at 96 or 120 h did not suppress but rather reduced oocyst production.     

Antigenic variation among strains of Eimeria maxima and E. tenella of the chicken.

The immunogenicity and cross-protection of Eimeria maxima or E. tenella in chickens against their homologous and heterologous strains were evaluated in two experiments. The immunizing strain of E. tenella protected against itself and partially against heterologous strains. The North Carolina (NC) strains of E. tenella were more virulent than the Delmarva (DMV) strains. Growth of the unimmunized groups was depressed 66% and 32% for the NC and DMV strains, respectively. Growth of the immunized-challenged groups (DMV and NC) was depressed by 13%. The DMV E. maxima strains were more virulent than the NC strains. Growth of the unimmunized challenged groups was depressed by 47% (DMV) and 13% (NC). Results demonstrated that there are antigenic variations among strains of two species of chicken coccidia.     

Development of protective immunity against Eimeria tenella and E. acervulina in White Leghorn chickens inoculated repeatedly with high doses of turkey coccidia

Repeated inoculation (immunization) of 2-week-old white leghorn chickens with 10(6) oocysts of the turkey coccidia Eimeria adenoeides or E. meleagrimitis partially protected chickens against moderate challenge with E. tenella or E. acervulina oocysts, but not with E. necatrix oocysts. After challenge, mean weight gains of the immunized chickens and the unchallenged controls did not differ significantly, but weight gains of unimmunized chickens were significantly lower. The mean feed-conversion ratio of the immunized challenged chickens was 3.14, as compared with 4.42 for unimmunized challenged control chickens. In general, immunization did not markedly reduce intestinal lesions. Repeated inoculation of chickens with the turkey coccidium E. gallopavonis failed to produce statistically significant protection against challenge with E. tenella, E. acervulina, or E. necatrix, as determined by weight gain, feed-conversion efficiency, and lesion scores. Antibody profiles of individual chickens did not correlate with protection.     

Potential of a recombinant antigen as a prophylactic vaccine for day-old broiler chickens against Eimeria acervulina and Eimeria tenella infections.

A genetically engineered Eimeria tenella antigen (GX3262), produced as a fusion protein with beta-galactosidase and identified with a monoclonal antibody, induced partial but significant protection in young broiler chickens against experimental E. tenella and Eimeria acervulina infections. The antigen appears to share a T-helper cell epitope with the parasite as evidenced by (a) booster inoculation with either the recombinant antigen or with a small number of live oocysts enhanced the protective immunity in GX3262 primed chickens, and (b) ability of the antigen to induce in vitro stimulation of T-cells from chickens immunized with antigen or parasite. These observations suggest the feasibility of a single vaccination of 1 or 2-day-old broilers with GX3262 to induce an acceptable degree of protective immunity. The implications of the observations reported here are far reaching in terms of a practical coccidiosis vaccine for poultry, and show for the first time that 1-day-old broiler chickens can be efficiently vaccinated with a recombinant antigen against one or more species of Eimeria.     

Comparative analysis of evaluation parameters in E. acervulina,E. maxima and E. tenella-infected broilers

Three parameters, body weight gain (BWG), intestinal lesion score (LS) and fecal oocyst shedding, were compared in broilers infected with major parasitic species; Eimeria acervulina, E. maxima, and E. tenella. First, two- and three-week-old chickens with Eimeria infection showed LS of approximately 3, but two-week-old chickens were more correlated with BWG. Second, significant differences in BWG were observed between male and female broilers challenged with Eimeria. Finally, E. maxima-infected broilers among three Eimeria species showed a higher relationship between BWG and LS, suggesting three considerations such as genders, age and Eimeria species for Eimeria experiments.     

Distribution of oocysts, sporocysts and sporozoites of Eimeria tenella and Eimeria maxima in the digestive tract of chicken.

The distribution of oocysts, sporocysts and sporozoites of Eimeria tenella and Eimeria maxima in the digestive tract of chicken and in excreta was investigated. At 1 h after the oral inoculation of E. tenella oocysts, the number of sporocysts in the cecum was 3.4 x 10(6) and decreased gradually thereafter, and the number of sporozoites in the cecum increased and remained at a high level until 12 h after the inoculation. Small numbers of sporocysts and sporozoites of E. tenella were found in other intestinal sites. A great number of E. maxima sporozoites was found, especially in the jejunum, 2 h after the inoculation. The findings that the largest populations of sporozoites of E. tenella and E. maxima were found in the cecum and the jejunum, respectively, indicate that the site specificity of sporozoite invasion for each species is determined before the invasion takes place.     

Pharmacodynamic evaluation of a reference and a generic toltrazuril preparation in broilers experimentally infected with Eimeria tenella or E. acervulina

1. The aim of this study was to investigate the effects on pigmentation, faecal oocyst output, immune responsiveness and reactive oxygen species (ROS) generation following treatment with either the reference toltrazuril (Baycox) or a generic preparation (gen-TTZ), during an experimental Eimeria tenella (Et) or E. acervulina (Ea) infection of 210 Ross broiler chickens.

2. Results showed a significant difference on the anticoccidial efficacy 6 d after treating infected animals with Baycox (Et: 99.69% and Ea: 99.52%) or gen-TTZ (Et:85.71% and Ea 81.81%).

3. Gen-TTZ-treated animals were less strongly carotenoid-pigmented than Baycox-treated broilers. Mean plasma carotenoid concentrations were significantly higher in groups treated with Baycox than in broilers given gen-TTZ.

4. Treatment of animals with Baycox led to a significant decrease in ability of the peripheral blood mononuclear cells to produce ROS in contrast to gen-TTZ-treated groups. Baycox, but not generic toltrazuril, increased IL-10 and decreased tumour necrosis factor alpha (TNF-α) concentrations in chickens infected with E. tenella and E. acervulina.

5. It is suggested that differences in anticoccidial efficacy may be observed when using a generic toltrazuril product. Hence, in addition to plasma profiles of drugs, standardised clinical control tests may be necessary for generic formulations, particularly if other parameters are important to achieve a better control of coccidiosis.