Inter- and intra-strain variation and PCR detection of the internal transcribed spacer 1 (ITS-1) sequences of Australian isolates of Eimeria species from chickens

The objective of this study was to confirm the presence of seven species of Eimeria involved in chicken coccidiosis in Australia by comparing internal transcribed spacer 1 (ITS-1) sequences, ITS-1 polymerase chain reaction (PCR) methods and to apply phylogenetic analysis to assess evolutionary relationships of Australian isolates. Twenty-two distinct ITS-1 regions of 15 Australian Eimeria isolates were sequenced, and analysed using maximum parsimony, distance and maximum likelihood methods. Poor bootstrap support, resulting from high ITS-1 sequence heterogeneity between all species groups, resulted in polychotomy of the Eimeria species in all three trees generated by these analyses. Percentage identity analyses revealed two distant ITS-1 lineages in both E. mitis and E. maxima at the same levels that separate the two species E. tenella and E. necatrix. One E. maxima lineage consisted of Australian isolates, the other American isolates, with one European sequence (originating from the same isolate) in each lineage. One Australian E. praecox sequence was only distantly related (33% variation) to three E. praecox sequences from Australian and European isolates. Short and long ITS-1 variants were isolated from both E. tenella (cloned line) and E. necatrix isolates with deletions (106 and 73 bp, respectively) in the short variants within the 3' region of the ITS-1 sequence. ITS-1 sequences of strains of both E. brunetti and E. acervulina species varied the least. Apart from E. maxima, all of the ITS-1 sequences of the six remaining individual species clustered to the exclusion of other species in all phylogenetic trees. Published ITS-1 tests for E. necatrix, E. acervulina, E. brunetti and E. tenella, combined with three new tests for E. mitis, E. praecox and Australian E. maxima amplified all respective Australian isolates specifically in a nested format using conserved ITS-1 PCR products as template to improve the sensitivity. All PCR tests were confirmed against a collection of 24 Australian chicken Eimeria isolates and contaminating species were detected in some instances. In conclusion, once the genetic variation between species and strains is determined, the ITS-1 is a good target for the development of species-specific assays, but the ITS-1 sequences alone do not seem suitable for the confirmation of phylogenetic inferences for these species. This study reports the first attempt at the analysis of the phylogeny and sequence comparison of the Eimeria species involved in chicken coccidiosis in Australia.     

Study on Coccidiosis of Scavenging Indigenous Chickens in Central Ethiopia

An investigation was made into coccidiosis of 190 scavenging indigenous chickens between September 2000 and April 2001 in three selected agroclimatic zones, in central Ethiopia. This was done through clinical, postmortem and microscopic examinations. Data were processed by chi-square and Mantel-Haenzel test. The study indicated that 25.8% (49/190) of the chickens were infected with coccidiosis and found to harbour one to four different species of Eimeria. Of these infected chickens, 30 (15.8%) and 19 (10.0%) were positive for clinical and sub-clinical coccidiosis, respectively. There was a significant altitude difference (chi2 = 14.7, p <0.001) in coccidiosis prevalence: 42.2% in chickens from highland region followed by 21.5% in mid-altitude and 13.1% in low-altitude areas. When quantified, the prevalence of coccidiosis was 2.66 and 4.83 times higher in the high-altitude than in mid-altitude (odds ratio, OR = 2.66, p<0.05) and low-altitude (OR = 4.83, p<0.001) chickens. The pathogenic Eimeria species responsible for clinical coccidiosis were E. necatrix, E. acervulina, E. maxima and E. tenella. With increasing demand for poultry products in developing countries, knowledge of production constraints in traditional management practices could help devise control strategies for constraints on backyard poultry production systems.     

Cloning and nucleotide sequencing of the second internal transcribed spacer of ribosomal DNA for three species of Eimeria from chickens in Taiwan

Coccidiosis of chickens caused by protozoan parasites of the genus Eimeria (Coccidia: Eimeriidae) is an enteric disease that results in great economic losses throughout the world, including Taiwan. Using polymerase chain reaction (PCR) with primers specific for the second internal transcribed spacer (ITS-2) of ribosomal DNA (rDNA), three species of Eimeria, E. tenella, E. maxima, and E. acervulina have been successfully characterised from chickens in Taiwan. The sizes of PCR products from various isolates representing these three species were between 370 and 580 base pairs (bp). After cloning and sequencing of the PCR products, high nucleotide sequence identity (96.8-100%) was observed within a species. In addition, ITS-2 nucleotide sequences for E. tenella had higher homology (98.5-99.3%) than E. maxima (81.6-96.5%) when compared with appropriate sequences deposited in GenBank. To our knowledge, this is the first report of a 412-bp ITS-2 sequence for E. acervulina from chickens.     

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.     

PCR及相关技术在鸡球虫种株鉴别方面的应用

鸡球虫病是危害养禽业的重大疫病之一,其病原属于艾美耳属(Eimeria)的球虫,世界上公认有7种。过去,其病原的鉴别一直是研究的难点。近年来,由于分子生物学技术,尤其是PCR及其相关技术的迅猛发展及在寄生虫分类中的应用,鸡球虫种、株鉴别方法也由以形态特征和生活史等为标准的传统分类学进入了以核酸为材料的现代分子分类学时代。文章对PCR及其相关技术在鸡球虫种、株鉴别方面的应用做了较为全面的阐述,并分析了其优缺点。     

Control of coccidiosis in chickens by vaccination

Control of coccidiosis in chickens has relied upon managerial measurements and the prophylactic use of coccidiostatic drugs. With the emergence of Eimeria strains that are resistant to these drugs the use and number of commercially available vaccines has increased. In this review various aspects that contribute to the development of coccidiosis are discussed, and an overview of the currently marketed coccidiosis vaccines is presented. Three groups of vaccines can be distinguished based on the characteristics of the Eimeria species included in the products: vaccines based on live virulent strains, vaccines based on live attenuated strains, and vaccines based on live strains that are relatively tolerant to the use of ionophores. These latter vaccines combine the early protective effect of ionophore treatment with the late protective effect of vaccination. The impact of future developments such as recombinant-DNA vaccines and changes in consumer's attitude towards broiler production are discussed.     

鸡球虫病疫苗的研究进展

鸡球虫病是由艾美耳球虫感染鸡引起的一种危害极其严重的寄生原虫病。目前,控制球虫病主要还是依赖于药物,但由于球虫耐药株的频繁出现和人们对药物残留等问题的关注,使人们更加注重探讨从免疫预防的角度去控制球虫病。本文综述了鸡球虫病活疫苗(强毒疫苗和弱毒疫苗)的研究和应用现状及重组疫苗(DNA疫苗和重组蛋白疫苗)的研究进展,同时介绍了新型疫苗佐剂的研究情况。     

Study on Coccidiosis in Kombolcha Poultry Farm,Ethiopia

A study on the occurrence of coccidiosis and distribution of Eimeria species in dead chickens 1-60 days of age, at Kombolcha Poultry Multiplication and Research Center (KPMRC), Ethiopia was conducted from November 2002 to April 2003. Out of the 965 dead birds, 370 (38.34%) were found to have clinical coccidiosis. The Eimeria species identified in this study were Eimeria brunetti, E. tenella, E. acervulina and E. necatrix with prevalence rates of 45.3%, 40.8%, 9.7%, and 4.1%, respectively. In the current study, E. brunetti was reported for the first time in Ethiopia. It was noted that clinical coccidioisis was more prevalent in those between 5 and 6 weeks of age. A statistically significant difference (p < 0.05) was observed in clinical coccidiosis prevalence among the different age groups studied. Various managerial problems that are associated with this high prevalence of clinical coccidiosis are identified and appropriate control strategies are recommended.     

福建省鸡球虫病感染情况的调查

为掌握福建省鸡球虫病的发病状况及影响因素,2009年12月至2010年11月,采用粪便漂浮法和卵囊培养法对本省的球虫病情况进行调查,并在实验室对送检的1 500份粪便样品进行了分析.结果发现当地鸡体内有6种球虫,分别是柔嫩艾美耳球虫(Eimeria tenlla)、巨型艾美耳球虫(E.maxima)、堆形艾美耳球虫(E...     

Interleukin-2 production in SC and TK chickens infected with Eimeria tenella

SC and TK inbred chicken strains display differential protective immunity to coccidiosis, SC being more resistant and TK susceptible to disease. In this study, the association between interleukin (IL)-2 and disease phenotype was assessed by cytokine quantification in serum, duodenum, cecum, and spleen cell cultures of SC and TK chickens experimentally infected with Eimeria tenella. In general, after primary infection, SC and TK strains produced equivalent amounts of IL-2 in all sources examined. However, after secondary infection, SC animals displayed significantly greater IL-2 levels in serum and the duodenum compared with strain TK. IL-2 production after reinfection with Eimeria may be an important factor contributing to the genetic differences in coccidiosis between SC and TK chickens and provides a rational foundation for cytokine-based immunotherapeutic approaches to disease control strategies.     

Molecular identification of Eimeria species infecting market-age meat chickens in commercial flocks in Ontario

A previously described multiplex PCR was evaluated for the identification and prevalence of Eimeria species in market-age commercial chicken flocks in Ontario. The multiplex PCR based on species-specific RAPD-SCAR markers was able to distinguish six available laboratory strains of Eimeria species (E. tenella, E. maxima, E. necatrix, E. mitis, E. acervulina, and E. brunetti) and E. tenella, E. maxima and E. acervulina in unknown field samples, including multiple infections in single reactions. No backyard (0/77) and 20/360 market-age commercial chickens were oocyst-positive using standard fecal flotation methods. PCR identified E. tenella alone (9/360, 2.5%), E. maxima alone (5/360, 1.38%), E. maxima plus E. tenella (5/360, 1.38%) and E. acervulina alone (1/360, 0.27%) in market-age commercial broilers. This is probably the first time the multiplex PCR has been evaluated in poultry establishments in Canada and illustrates the value of the tool in coccidiosis epidemiology on commercial farms.     

鸡球虫病弱毒疫苗的免疫机理及研究进展

鸡球虫病是由艾美耳属球虫引起的一种危害性极其严重的寄生原虫病,呈世界性分布,是最主要的鸡病之一。目前主要用抗球虫药物对其进行防制,但随着耐药虫株的产生和药物残留对肉禽品质及人体健康的影响等问题的出现,对鸡球虫活疫苗的研究成为研究热点。作者对鸡球虫弱毒疫苗的免疫机理及其鸡胚传代致弱方法、理化致弱方法和早熟选育致弱法的研究进展作一概述,并对鸡球虫弱毒疫苗的应用前景进行了展望。     

斯氏艾美耳球虫ITS1-5.8S rRNA-ITS2序列的克隆及PCR检测方法的建立

通过对多种鸡球虫和松鼠球虫18SrRNA和28SrRNA进行序列比对分析,在18SrRNA 3′端和28SrRNA 5′端保守区设计艾美耳属通用引物,以斯氏艾美耳球虫洛阳分离株LY卵囊基因组DNA为模板首次成功克隆到斯氏艾美耳球虫完整的ITS1-5.8SrRNA-ITS2序列,其大小为1 178bp,其中ITS1序列长度为423bp,5.8SrRNA为155bp,ITS2为600bp,斯氏艾美耳球虫LY株ITS1/2序列高度变异,与鸡球虫、啮齿动物球虫的序列相似性低于60%。然后在斯氏艾美耳球虫ITS1/2序列超变区设计种特异引物,建立了灵敏、特异的PCR检测方法。本研究结果将为兔球虫强致病种的临床诊断和揭示兔球虫种群遗传特征提供有效的分子工具。     

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.     

Differential diagnosis of five avian Eimeria species by polymerase chain reaction using primers derived from the internal transcribed spacer 1 (ITS-1) sequence

Chicken coccidia are protozoan parasites of the genus Eimeria. They cause economical losses in the poultry industry globally. The various species can be distinguished on the basis of the morphology of the oocysts and parasitic site in intestine, but these criteria sometimes are unreliable. Therefore, a species-specific polymerase chain reaction (PCR) was developed. Based on variable sequence regions, specific primers were constructed for the differentiation of five Eimeria species (Eimeria acervulina, E. brunette, E. maxima, E. necatrix, and E. tenella). PCR products were amplified from coccidian vaccine (coccivac-D and coccivac-B) and E. tenella and were subsequently sequenced. Similarities of the five species sequences between the vaccines and Genbank were 94-100%. Analysis of the E. tenella internal transcribed spacer 1 (ITS-1) partial sequence from Taiwan and from Genbank indicated that the similarity was 99.6%. The PCR sensitivity test of E. tenella in Taiwan is 50 oocysts. The five sets of primers will not amplify any non-specific bands of the chicken genome or its intestinal contents. Therefore, the five sets of specifically designed primers are guaranteed to be useful for differential diagnosis of avian coccidiosis caused by Eimeria spp.     

基于ITS1-5.8S rRNA-ITS2序列兔肝球虫PCR检测方法的建立

通过对多种鸡球虫和松鼠球虫18S rRNA和28S rRNA进行序列比对分析,在18S rRNA 3’端和28S rRNA 5’端保守区设计艾美耳属通用引物,以斯氏艾美耳球虫洛阳分离株LY卵囊基因组DNA为模板首次成功克隆到斯氏艾美耳球虫完整的ITS1-5.8S rRNA-ITS2序列,其大小为1178bp,其中ITS1序列长度为423bp,5.8S rRNA为155 bp,ITS2为600 bp,斯氏艾美耳球虫LY株ITS1/2序列高度变异,与鸡球虫、啮齿动物球虫的序列同源性低于60%。然后在斯氏艾美耳球虫ITS1/2序列超变区设计种特异引物,建立了灵敏、特异的PCR检测方法。本研究结果将为兔球虫强致病种的临床诊断和揭示兔球虫种群遗传特征提供有效的分子工具。     

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.     

陕西省杨陵区鸡球虫病原种类的调查研究

对杨陵区四乡一镇的６８组鸡群进行了鸡球虫病的调查研究。结果表明，杨陵区鸡球虫病比较普遍，１５￣５０日龄雏鸡的球虫病特别严重。全区鸡的球虫平均感染率为５８．３％，在所检查的青年，成年鸡群中，笼养鸡群的球虫感染率为３５．１％，散养鸡群的球虫感染率为５７．４％。雏鸡的球虫感染率为６８．４％。经实验室鉴定，共查见柔嫩艾美耳球虫，毒害艾美耳球虫，巨型艾美耳球虫，堆型艾美耳球虫和缓艾美耳球虫和哈氏艾美耳球虫等     

The efficacy and economic benefits of Supercox, a live anticoccidial vaccine in a commercial trial in broiler chickens in China

The efficacy and economic benefits of Supercox, a live anticoccidial vaccine were examined and compared with an anticoccidial drug in a trial in broiler chickens under modern commercial conditions in China. In total, 40,660 chickens were used in the present study, half of which were vaccinated with the Supercox vaccine comprising a precocious line of Eimeria tenella and non-attenuated lines of Eimeria maxima and Eimeria acervulina, and the other half were medicated with Diclazuril delivered as feed additive at the dosage of 1mg/kg of feed. The vaccine was administered orally to 7-day-old chickens. No clinical diseases were diagnosed in any of the vaccinated birds. However, clinical coccidiosis occurred in a large proportion of medicated control birds and these chickens had to be treated with anticoccidial drugs (Diclazuril and Toltrazuril). Comparison of production performance between vaccinated birds and medicated control birds revealed that the vaccine Supercox performed better than anticoccidial drugs in terms of mortalities, costs and overall economic benefits (profits). These findings demonstrated that the use of the Supercox vaccine could control clinical coccidiosis in broilers and achieve production performance superior to that using anticoccidial drugs, particularly where drug resistance might result in failure to control clinical diseases.     

Responses of chickens vaccinated with a live attenuated multi-valent ionophore-tolerant Eimeria vaccine

Coccidiosis, caused by Eimeria species, is a serious economic disease of chickens (Gallus gallus) and the search for vaccines to control the disease is intensifying especially with the increasing threat of drug resistance. A live attenuated multi-valent ionophore-tolerant Eimeria vaccine has been developed that contains three ionophore-resistant Eimeria species, E. tenella, E. maxima and E. acervulina. The attenuated lines were derived from virulent field strains resistant to monensin ionophore by selection for early development in chicks. The vaccine was administered by gavage and through drinking water to broiler chickens, Chinese Yellow strain, reared in wire cages. Vaccinated medicated birds performed better than vaccinated unmedicated and medicated unvaccinated groups. The final mean weights of vaccinated medicated birds were significantly higher (P<0.05), and a better vaccine protection index, using both vaccinating methods, was achieved. Results indicated that concomitant use of ionophores and vaccines could be a useful adjunct to planned immunization in the control of coccidiosis.