Application of polymerase chain reaction based on ITS1 rDNA to speciate Eimeria

A method was developed to recover Eimeria spp. oocysts directly from poultry litter and determine which species of Eimeria were present using polymerase chain reaction (PCR) based on the ITS1 rDNA sequence. The species composition of Eimeria oocysts was also compared before and after propagation in susceptible chickens to determine if the relative proportion of each species changed after expansion. In samples from two broiler operations, ITS1-PCR was able to detect Eimeria spp. oocysts recovered from litter, with Eimeria acervulina, Eimeria maxima, and Eimeria praecox being the predominant species present therein. Although Eimeria tenella was found in one sample, the other species--Eimeria brunetti, Eimeria necatrix, and Eimeria mitis-were not detected. The species composition as determined by ITS1-PCR did not appear to appreciably alter after expansion in susceptible chickens. The described method represents a rapid means for determining the major Eimeria species in a poultry operation and may be helpful in choosing a particular live oocyst vaccine formulation to protect chickens against coccidiosis.     

Diagnosis of Eimeria species using traditional and molecular methods in field studies

The objective of this study was to identify and characterize species of Eimeria in broiler chickens using traditional morphological and pathological plus molecular (DNA amplification) diagnostic methodologies. Using a combination of those techniques it was possible to identify the presence of multiple circulating species in the flock as well as higher frequencies for some of them, especially Eimeria praecox and Eimeria maxima, which were identified in 100% of the flocks. The frequencies of the other species were Eimeria mitis and Eimeria necatrix (93.3%), Eimeria tenella (76,7%), Eimeria acervulina (56.7%) and Eimeria brunetti (16.7%). However using the lesion score, the most common species were E. maxima (46.7%), E. acervulina (30%), E. tenella (23.3%), and E. necatrix (10%). E. brunetti and E. praecox were not identified by using lesion score. DNA amplification had detection sensitivity for Eimeria species in the field samples of at least 20 oocysts. The implementation of DNA amplification as a routine diagnostic technique in aviaries can assist Eimeria population.     

布氏艾美耳球虫南宁株的分离及鉴定

本试验从南宁市郊区某鸡场感染球虫病的病鸡粪便中收集到混合卵囊,采用改良过的琼脂单卵囊分离法从中分离纯化出2个球虫虫株,并对其中的1个球虫分离株进行了鉴定,鉴定结果为:该株球虫卵囊呈椭圆形或卵圆形,卵囊平均大小为(22.51±0.4790)μm×(15.94±0.5599)μm,平均卵型指数为1.41212±0.06714,裂殖体平均大小为31.33 μm×27.46 μm,潜在期为120 h 50 min,排卵高峰期为感染后第6天,最短孢子化时间为18 h 50 min,寄生部位为小肠中、下段,属中等毒力的球虫虫株。通过将这些测定和观察到的指标与各类文献中所记载的各种鸡艾美耳球虫的特征进行了对比,经过综合分析后,将该分离株球虫鉴定为布氏艾美耳球虫,并且将它命名为布氏艾美耳球虫南宁株,记为Eimeria.brunetti-GXNN。对该虫株的成功分离鉴定,为进一步了解本地球虫虫株的生物学特性,开展鸡球虫病的免疫预防奠定了基础。     

布氏艾美耳球虫保定株的鉴定及致病性研究

本研究采用琼脂薄膜球虫单卵囊分离技术,从河北省保定市暴发球虫病的某鸡场鸡粪便中成功分离出布氏艾美耳球虫卵囊。生物学特性研究结果表明,该虫种潜隐期为125 h,卵囊大小为26.1 μm×21.3 μm,形状指数为1.22,寄生与病变部位主要在小肠后段、直肠和泄殖腔,特征性病变是肠黏膜层有出血斑纹及血性内容物。感染一定剂量该球虫,鸡的生产性能受到影响,当感染剂量为2×10⁵个/羽时会出现鸡死亡,表明该虫株具有较强致病性;应用特异PCR方法对获得的卵囊进行分子鉴定,结果确认所分离和纯化的球虫卵囊为布氏艾美耳球虫纯种,并将该虫株命名为布氏艾美耳球虫保定株--BBD株。     

Immunohistochemical study of S100-like protein in Eimeria brunetti and Eimeria acervulina

We have investigated the expression of a calcium-binding protein, the S100 protein, in Eimeria brunetti and Eimeria acervulina stages. For this purpose, paraffin sections of distal ileum and bursa of Fabricius or duodenum from experimentally infected chickens were treated with anti-alpha-S100 (anti-alpha subunit of S100 protein) and anti-beta-S100 (anti-beta subunit of S100 protein) monoclonal antibodies and anti-S100 whole molecule polyclonal antibody. The avidin-biotin peroxidase method was used to demonstrate immunoreactivity. In the ileum, our results reveal a positive immunoreaction for the beta subunit and S100 whole molecule within the macrogametes of E. brunetti, whereas they were devoid of immunostaining after treatment of the paraffin sections with the anti-alpha-S100 antiserum. Schizonts and oocysts of E. brunetti and all the E. acervulina stages gave a negative reaction after treatment with any of the three antiserum used in the study. This result indicated that the S100 protein molecules within these stages were not recognized by the antibodies, suggesting that these molecules are different from those identified in macrogametes of E. brunetti. By contrast, in the epithelial cells, lining the lumen of the bursa of Fabricius, macrogametes of E. brunetti were stained by the three antibodies used. These results may indicate the existence of metabolic adaptations that enable the parasite to invade tissue sites different from those where the parasite usually develops.     

Efficacy of decoquinate against drug sensitive laboratory strains of Eimeria tenella and field isolates of Eimeria spp. in broiler chickens in China

The efficacy of decoquinate against Eimeria infections in broiler chickens was evaluated using two drug sensitive laboratory strains of Eimeria tenella and 20 field isolates of Eimeria spp. collected from farms in China where various anticoccidials (including maduramicin) had been used. Decoquinate (20-40 ppm in feed) and maduramicin (5 ppm) were efficacious against E. tenella laboratory strains, but decoquinate more so than maduramicin. Body weight gains of E. tenella infected chickens were significantly improved, and caecal lesions were prevented, by feeding either decoquinate or maduramicin. Decoquinate also prevented oocyst production, but maduramicin did not. Most (18/20) Eimeria field isolates were resistant to maduramicin, judged by oocyst production; decoquinate at > or =20 ppm completely controlled all 20 field isolates. Decoquinate has potential value as a broiler anticoccidial in China and other countries where it has not been previously used.     

Quantitative real-time PCR assays for detection and quantification of all seven Eimeria species that infect the chicken

The development and validation of real-time quantitative PCR (qPCR) assays specific to all seven Eimeria species that cause coccidiosis in the chicken is described. The presented work utilizes previously published assays for Eimeria maxima, E. necatrix and E. tenella and adds assays for E. acervulina, E. brunetti, E. mitis and E. praecox. These assays target unique single copy sequences derived from sequence characterized amplified region (SCAR) markers. All seven qPCR markers were sequenced from multiple strains and confirmed to be non-polymorphic and identical to the original SCAR sequence. Sequences conserved within each species were chosen with the aim of developing genuinely universal markers, providing global coverage. An exact match for the primers and TaqMan(?) probe during PCR cycling enables precise relative quantification of multiple species in a mixture regardless of the strains present. All markers utilized in these qPCR assays are absolutely species-specific and support reproducible quantification across a wide linear range, unaffected by the presence of non-target species or other contaminating DNA. The sensitivity of these assays indicates that DNA equivalent to a single sporulated oocyst can be consistently detected. These assays will be a valuable tool from both industry and research perspectives. Comparison of our panel of qPCR assays with results derived by microscopy, the traditional Gold Standard, using poultry farm field samples support their efficacy.     

Isolation and pathogenicity of Australian strains of Eimeria praecox and Eimeria mitis

Objective To determine the presence of E praecox and E mitis in Australia, to isolate representative strains of these species from chickens and determine their pathogenicity.
Design Morphological, physiological and cross protection studies were undertaken to confirm the identity of Australian isolates of E praecox and E mitis.
Procedure Oocysts were isolated from a backyard flock at Jimboomba, southeastern Queensland and numbers of E praecox and E mitis enriched by passage in chickens immune to five other species of poultry Eimeria . Oocysts of mean conformation and size of the two species were purified by single oocyst passage. Two isolates that closely matched recorded parameters for E praecox and E mitis were selected and designated JP and JM respectively. The cross protection between the isolates and E acervulina was determined by infection and challenge experiments. The virulence of the two isolates was determined by comparing weight gains of groups of birds inoculated with JP isolate or JM isolate with untreated groups.
Results Isolates JP and JM most closely matched recorded parameters of E praecox and E mitis respectively. Groups of chickens, previously infected with JP and JM isolates, showed no significant protection against infection with E acervulina . In a separate trial, groups of susceptible chickens inoculated with 10⁵ oocysts of JP and JM isolates showed significantly reduced weight gains compared with untreated controls.
Conclusion Isolates JP and JM are E praecox and E mitis respectively, confirming the presence of these species in Australia. These isolates were found capable of causing significant reductions in weight gains in susceptible chickens.     

柔嫩艾美耳球虫广西南宁株的分离及鉴定

利用从南宁市郊养鸡场球虫病鸡粪便中收集的球虫混合种卵囊感染小鸡,再应用单卵囊分离感染技术,从感染鸡盲肠中收集的卵囊分离纯化获得1株纯种球虫,经鸡体传代增殖,对该虫株的卵囊大小和卵形指数、潜在期、排卵高峰期、最短孢子化时间、寄生部位、致病性等指标进行观察和测定。结果测得该虫株卵囊的平均大小为(25.743±1.94126)μm×(21.4±1.85985)μm,平均卵型指数为1.2067±0.07;潜在期为140 h;其排卵囊峰期在第6～9天,最高峰在第7天;最短孢子化时间为19 h;寄生部位在盲肠;对两周龄的艾维茵鸡,当使用5×104的孢子化卵囊感染剂量时死亡率为7.5%。根据这些测定和观察到的指标综合鉴定该分离株球虫为柔嫩艾美耳球虫,并命名为柔嫩艾美耳球虫广西南宁株(Eimeria.tenella-GXNN),该研究结果为进一步研究本地区鸡球虫病的药物治疗和免疫预防等奠定了基础。     

鸡巨型艾美耳球虫单卵囊分离及雏鸡扩增试验

采用饱和食盐水漂浮法收集就诊病死球虫阳性鸡小肠中段内容物中的球虫卵囊,恒温培养至孢子化后,用2%琼脂薄板进行球虫单卵囊分离,分别感染7只5日龄雏鸡,对据形态学鉴定为巨型艾美耳球虫的分离株进行2代单卵囊分离和雏鸡感染,取其后代以每只1.0×10⁴个卵囊感染10只10日龄雏鸡进行卵囊扩增,获得大量纯种巨型艾美耳球虫卵囊。结果表明,刀片切割琼脂薄板单卵囊分离法操作简便,单卵囊感染成功率较高,准确率达100%,适用于纯种卵囊的分离与扩增。     

33个小型鸡场球虫流行病学调查

对天津、河北、山东等地33个小型养鸡场做了球虫病原学流行调查。根据卵囊形态，最短潜在期．裂殖体部位和大小，病变等具有鉴别意义的特征做综合鉴定。结果显示，33个鸡场都有球虫的感染。共鉴定出7个种．其中有28个鸡场有堆形艾美耳球虫(E．acervulina)，占84．84％．26个场有柔嫩艾美耳球虫(E．tenella)．占78．78％．25个场有巨型艾美耳球虫(E．maxima)，占75．8％，早熟艾美耳球虫(E．praecox)，和缓艾美耳球虫(E．mitis)，布氏艾美耳球虫(E．brunetti)和毒害艾美耳球虫(E．necatrix)各1个场有．各占3％。只有一个鸡场单独感染一种球虫．其余都是混合感染2种或2种以上球虫，其中有2个鸡场混合感染4种球虫。     

Eimeria praecox infection ameliorates effects of Eimeria maxima infection in chickens

The effect of Eimeria praecox on concurrent Eimeria maxima infection was studied in susceptible chickens. Clinical signs of coccidiosis were assessed in single E. praecox or E. maxima infections and compared to dual infection with both Eimeria species. Groups infected solely with 10(4)E. maxima oocysts displayed weight gains that were 48% of weight gain in uninfected controls. Weight gain in chickens infected only with 10(4)E. praecox oocysts was 90% of uninfected controls. Average weight gain in chickens infected with both E. maxima and E. praecox was 79% of controls, and showed no significant difference (P>0.05) from weight gain in E. praecox-infected chickens. Feed utilization (feed conversion ratio, FCR) in chickens infected with both species showed no significant difference (P>0.05) from FCR in non-infected controls or chickens infected with E. praecox alone; all showing a significant difference (P<0.05) from FCR in chickens infected solely with E. maxima. Although E. praecox did not appear to have a negative effect on weight gain and FCR, it did cause a significant decrease in serum carotenoids. Analysis of oocysts excreted by chickens during dual infection showed little effect of E. praecox on E. maxima oocyst production.     

Relative value of oocyst counts in evaluating anticoccidial activity.

Birds medicated with roxarsone and in another experiment with zoalene in the feed produced higher oocysts counts than unmedicated control birds receiving the same oocyst dose of Eimeria tenella or a mixture of six species (E. tenella, E. necatrix, E. brunetti, E. maxima, E. acervulina, and E. mitvati). These experiments confirm the conclusion that oocyst counts constitute an unsatisfactory and unreliable parameter for judging effectiveness of an anticoccidial even though such increases are a relatively rare occurrence in anticoccidial evaluation experiments.     

Anticoccidial effect of green tea-based diets against Eimeria maxima

Anticoccidial effects of green tea (GT)-based diets were evaluated in chickens following oral infection with Eimeria maxima an ubiquitous intestinal parasite of poultry that impairs the growth and feed efficiency of infected birds. Five-week-old chickens were assigned to four groups (GT 0.5%, GT 2.0%, untreated/infected and non-infected control) and each group consisted of 15 chickens. Chickens were fed a standard diet supplemented with ground green tea for 2 weeks prior to infection with E. maxima (10,000 sporulated oocysts per bird). The effects of green tea on E. maxima infection were assessed by two parameters, fecal oocyst shedding and body weight gain. The green tea-fed chickens produced significantly reduced fecal oocysts (P<0.05) when compared to the E. maxima-infected group fed standard diet. The green tea-based diet, however, did not improve body weight loss caused by E. maxima infection. This study is the first to demonstrate anticoccidial effect of green tea on Eimeria parasites.     

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.     

特异PCR方法用于鸡的3种艾美耳球虫混合感染鉴别的研究

用单卵囊分离法获得的鸡的3种艾美耳球虫（每种各2株）卵囊：柔嫩艾美耳球虫（Eimeria tenella）、巨型艾美耳球虫（E．maxima）、堆型艾美耳球虫（E．acervulina）。经纯化、提取基因组DNA后，用报道的种特异引物做PCR扩增分析，以确定是否为纯种。结果发现这3种球虫均存在混合感染的情况。该结果为进一步研究这3种球虫奠定了基础，并说明特异PCR方法能够有效地、快速地鉴别球虫虫种。     

堆型艾美尔球虫的分离与病理学研究

采用单卵囊感染技术,从鸡球虫混合种中分离出一株堆型艾美尔球虫(Eimeria acenrulina).并对该株堆型艾美尔球虫的病理学做了进一步研究.利用堆型艾关尔球虫的寄生部位、肉眼病变、最小潜在期等生物学特性进行鉴定,并做回归试验,用此球虫卵囊1×105个感染雏鸡.取感染5d后有明显病理变化的十二指肠组织,采用石蜡切片技术,进行病理学观察.结果表明,虫体寄生于肠绒毛上皮细胞内,大量的肠绒毛遭到破坏、断裂和脱落.     

Isolation of five species ofeimeria from chickens in Bangladesh

Five species of Eimeria, namely E. acervulina, E. tenella, E. maxima, E. brunetti and E. necatrix were identified in chickens in Bangladesh on the basis of lesions seen at post-mortem examinations of naturally infected birds, and on the dimensions of oocysts and the lesions seen in chicks experimentally infected with single oocyst derived strains. The use of filter top, polycarbonate cages permitted the isolation of strains without sophisticated animal isolators and is appropriate for use in laboratories throughout the developing world. Responses to a questionnaire sent to all known intensive poultry farms suggested that coccidiosis is a major disease. For control, producers rely mostly on management procedures and the tactical use of sulphonamides; in-feed chemoprophylaxis is not widely used. These control measures are unsatisfactory and recent coccidiosis outbreaks were reported from seven of 16 farms. There was evidence of a seasonal incidence in clinical coccidiosis.     

5株柔嫩艾美耳球虫对4种抗球虫药的抗药性

选择260只19日龄雏鸡，随机分成26组，每组10只，研究柔嫩艾美耳球虫(E．tenella)南京株、凤阳株、扬州株、宣城株和蒙城株对地克珠利(Diclazuril)、马杜霉素(Maduramicin)、氯羟吡啶(C1opido1)、氯苯胍(Robenidine)的抗药性。每株球虫均设4个感染用药组、1个感染不用药组，另外设1个不感染不用药组。以POAA、RLS、ROP、ACI作为指标，判定5株球虫对4种药物的抗药性。结果表明凤阳株、扬州株和蒙城株分别仅对马杜霉素、氯苯胍、地克珠利敏感，南京株和宣城株对该4种药物均已产生不同程度的抗药性。提示目前在凤阳、扬州和蒙城地区可分别合理地使用马杜霉素、氯苯胍和地克珠利，对其它药物须减少或暂停使用。     

Characterisation and histopathological observations of a selected Brazilian precocious line of Eimeria acervulina

Precocious lines of Eimeria acervulina "Cu" and "I" strains were obtained after 25 passages of oocysts in chickens that showed a shortening of the prepatent period for first oocyst output from 96 h to 81 and 82 h, respectively. Both precocious lines were evaluated for pathogenicity using as criteria weight gain, lesion score and total oocyst production. Infection of the "Cu" precocious line in chickens showed a high weight gain, low lesion score and low oocyst production, when compared to parent strain infected chickens. However, the results did not show a significant difference in relation to the criteria used above for the E. acervulina "I" precocious line when compared to its parent strain. This suggests a low degree of attenuation for the "I" strain but good attenuation for the precocious "Cu" line. The histopathological observations of chickens infected with the E. acervulina "Cu" parent strain and precocious line, comparing life cycle and intestinal lesions, showed: (1) parasite stages only in the border cells of infected chicken intestinal villi, for the precocious line; (2) parasite stages in the border cells of the intestinal villi and submucosa cells near the Lieberkühn glands of the intestine; and (3) high degree of inflammatory cells around the parasites in chickens infected with the parent strain. The "Cu" strain was also characterized for sensitivity against eight anticoccidial drugs. Sensitivity was observed for four anticoccidial drugs and partial resistance for four other drugs, although the strain had never had previous contact with anticoccidial drugs, suggesting the presence of a natural resistance factor. This Brazilian E. acervulina "Cu" precocious line showed attenuation for pathogenicity in chickens, suggesting that it could be a suitable strain for use as a live vaccine in Brazil.