3种兔球虫ITS-1序列测定与系统发育分析

从河北省兔场分别单卵囊分离孢子化大型艾美耳球虫卵囊、黄艾美耳球虫卵囊及肠艾美耳球虫卵囊,接种无球虫兔后获得纯种卵囊,CTAB法提取孢子化卵囊基因组DNA。利用艾美耳属球虫18SrDNA和5．8SrDNA保守引物,PCR扩增3种兔球虫ITS-1片段,产物纯化后测序。将3种球虫ITS-1测序结果与GenBank发布的兔球虫ITS-1序列进行比对和遗传距离比较,绘制系统发育树。结果表明,大型艾美耳球虫、黄艾美耳球虫及肠艾美耳球虫河北株分别扩增出424、455、434bp的ITS-1片段。大型艾美耳球虫、黄艾美耳球虫及肠艾关耳球虫河北株与GenBank中发布的同种兔球虫ITS-1序列相似性分别为97．4％、97．9％和96．9％。系统发育树显示兔球虫ITS-1序列形成1个单系群,该单系群根据寄生部位分为2个姊妹群。     

3种兔球虫18S rDNA部分序列测定与系统发育分析

采用单卵囊分离法从河北某兔场分离大型艾美耳球虫、黄艾美耳球虫及肠艾美耳球虫,接种无球虫兔后获得大量纯种卵囊,CTAB法提取孢子化卵囊基因组DNA。利用艾美耳属球虫18S rDNA保守引物,PCR扩增3种兔球虫18S rDNA片段,产物纯化后测序。将3种球虫18S rDNA测序结果与GenBank中发布的兔球虫18S rDNA序列用DNAStar软件进行比对。使用MEGA4.0软件对兔球虫18S rDNA进行同源性比较,并绘制遗传进化树。结果表明,大型艾美耳球虫扩增出大小为1 521bp的18S rDNA片段;黄艾美耳球虫及肠艾美耳球虫均扩增出大小为1 520bp的18S rDNA片段。序列比对结果显示,3种河北株兔球虫与GenBank中相应的3种兔球虫18S rD-NA(EF694016、EF694011、EF694012)相似性分别为99.6%、99.6%和100%。3种河北株兔球虫序列和GenBank中兔球虫18S rDNA序列(EF694007-EF694017)位于一个单系集群。     

黄艾美耳球虫单卵囊分离及PCR鉴定

为了获得纯种黄艾美耳球虫,从张家口某兔场的兔粪中分离黄艾美耳球虫孢子化卵囊,单卵囊接种无球虫兔,以饱和盐水漂浮法收集子代卵囊,利用CTAB法提取黄艾美耳球虫卵囊基因组DNA,并根据Gen Bank中发表的艾美耳属球虫保守序列设计引物,PCR扩增ITS并测序,设计特异性引物鉴定黄艾美耳球虫。结果表明:黄艾美耳球虫ITS1序列长330 bp,5.8S r DNA序列长157 bp,ITS2序列长522 bp。在黄艾美耳球虫ITS1/2序列高变区设计种特异性引物,与大型艾美耳球虫和肠艾美耳球虫无交叉反应。说明建立的鉴定黄艾美耳球虫的PCR方法灵敏、特异。     

兔肠艾美耳球虫实时荧光定量PCR检测方法的建立

为建立一种肠艾美耳球虫定量的检测方法,在肠艾美耳球虫核糖体第二内转录间隔区(ITS2)序列设计一对特异性引物,以10倍倍比稀释的已知卵囊含量的肠艾美耳球虫DNA为模板进行SYBR GreenⅠreal-time PCR的扩增和标准曲线的建立。结果显示,最低可检测1个卵囊含量的样品,与同属的黄艾美耳球虫、中型艾美耳球虫、大型艾美耳球虫均不发生交叉反应,重复性变异系数小于2%。表明建立的实时荧光定量PCR检测方法灵敏度高,特异性强,重复性好,可为快速检测肠艾美耳球虫提供有效的方法。     

四种兔艾美耳球虫卵囊产量的比较

分别用4种兔艾美耳球虫经口接种45日龄无球虫感染兔,接种剂量为1×104个卵囊/兔。感染后4d～20d,以麦克马斯特氏法计数每天排出的卵囊。结果表明,肠艾美耳球虫排卵量最多,为365.76×106个,中型艾美耳球虫为317.26×106个、黄艾美耳球虫为304.36×106个,大型艾美耳球虫排卵量最少,为200.12×106个。大型艾美耳球虫在感染后第6.5天有卵囊排出,第10.5天达到高峰(66.50×106个/只),占总量的33.23%;肠艾美耳球虫在感染后第9天有卵囊排出,第13天达到高峰(138.40×106个/只),占总量的37.83%;黄艾美耳球虫在感染后第9天有卵囊排出,第11天达到高峰(102.80×106个/只),占总量的33.78%;中型艾美耳球虫在感染后第4.5天有卵囊排出,第6.5天达到高峰(167.8×106个/只),占总量的52.89%。     

福建省家兔球虫病感染情况调查

为掌握福建省家兔球虫病的发病情况及影响因素,采用饱和盐水漂浮法和卵囊培养法分别对福建省9县市的兔场进行了家兔球虫病感染情况的调查。结果表明,所调查的县市中家兔球虫病平均感染率为44.00%;幼兔的感染率较高,平均达59.33%,种兔的感染率相对较低,平均为4.71%;不同品种感染情况为福建黄兔感染率最高达51.52%,福建白兔较低为4.41%;种兔和幼兔的感染率2013年度分别为15.63%、89.26%明显高于2012年度的3.14%和32.85%;本次共检出12种艾美耳球虫,分别是斯氏艾美耳球虫、中型艾美耳球虫、大型艾美耳球虫、黄艾美耳球虫、新兔艾美耳球虫、野兔艾美耳球虫、纳格浦尔艾美耳球虫、无残艾美耳球虫、长形艾美耳球虫、穿孔艾美耳球虫、梨形艾美耳球虫、肠艾美耳球虫。     

柔嫩艾美耳球虫河北株致病性检测及ITS-1基因序列分析

为分析鸡柔嫩艾美耳球虫(Eimeria tenella)河北株的致病性及其ITS-1基因序列遗传变异特点,对临床分离的柔嫩艾美耳球虫河北株通过人工感染雏鸡试验验证其致病性,并计算其半数致死量(LD_(50)),采用RT-PCR对柔嫩艾美耳球虫河北株的ITS-1基因进行扩增、克隆,测序后进行生物信息学分析其基因序列变异情况。结果显示:柔嫩艾美耳球虫河北株对雏鸡有较强的致病性,其LD_(50)为3.16×10~4个/只;柔嫩艾美耳球虫河北株的ITS-1基因与GenBank登录的柔嫩艾美耳球虫ETSH4PF3-17株和柔嫩艾美耳球虫上海株的相似性在97.7%～99.0%之间,系统发育进化树分析显示柔嫩艾美耳球虫河北株与GenBank发表的柔嫩艾美耳球虫ETSH4PF3-17株和柔嫩艾美耳球虫上海株聚为一支,亲缘性最近,与其它虫株亲缘性较远;与GenBank发表的柔嫩艾美耳球虫ETSH4PF3-17株序列相比,柔嫩艾美耳球虫河北株的ITS-1基因序列中在第4、13、16、425位4个碱基发生缺失;第258、348位2个碱基发生变异,由C变为T,由G变为T。研究结果为进一步研究柔嫩艾美耳球虫河北株遗传变异情况提供参考依据。     

黄艾美耳球虫河北株18SrDNA部分序列测定及系统发育分析

为了进一步确定黄艾美耳球虫河北株虫种,试验根据GenBank中发表的黄艾美耳球虫18SrDNA基因序列设计引物,建立PCR方法对黄艾美耳球虫河北株基因片段扩增、测序,并进行系统发育分析.结果表明:扩增出大小为1 465bp的清晰条带;黄艾美耳球虫河北株18S rDNA序列测定结果与GenBank中的黄艾美耳球虫EF69...     

北京地区家兔球虫种类的初步调查

报道了北京周边5个地区家兔球虫种类的初步调查结果，初步认定了北京地区存在13个球虫种，即兔艾美耳球虫、无残艾美耳球虫、黄艾美耳球虫、盲肠艾美耳球虫、新兔艾美耳球虫、中型艾美耳球虫、小型艾美耳球虫、大型艾美耳球虫、斯氏艾美耳球虫、肠艾美耳球虫、梨形艾美耳球虫、松林艾美耳球虫、穿孔艾美耳球虫。     

鸡六种艾美耳球ITS-1的克隆和序列分析

为对上海地区分离的6种鸡艾美耳球虫内转录间隔区1(ITS-1)进行克隆和序列分析,探索ITS-1区域序列在鸡球虫分类学中的作用,利用一对属特异性引物,对上海地区分离的6种球虫的ITS-1序列进行PCR扩增,扩增产物克隆到pMD18-T后进行序列测定,并与GenBank上下载的序列进行系统进化树分析.结果显示,巨型艾美耳球虫有大小分别为547bp和422bp两条PCR扩增条带;和缓艾美耳球虫有大小为627bp和493bp两条PCR扩增条带;其他4种球虫均为一条PCR扩增条带,大小分别为柔嫩艾美耳球虫668bp,毒害艾美耳球虫691 bp,堆型艾美耳球虫507 bp,早熟艾美耳球虫541 bp.6种球虫序列之间的同源性为34%～52%.系统进化分析显示,各个种分别与GenBank中下载的对应球虫种在同一分支上.     

Sequence and Phylogenetic Analysis of rDNA ITS of Three Eimeria Species in Rabbit

In order to study whether the internal transcribed spacers (ITS) sequence could be used as a molecular marker for the species identification of rabbit coccidian, the rDNA ITS of Eimeria intestinalis, Eimeria flavescens and Eimeria magna were amplified by polymerase chain reaction (PCR), and were cloned into pGEM-T Easy vector subsequently. The positive recombinant plasmids were identified by PCR and then sequenced. By sequence comparison and comparative analysis with the relative sequences of rabbit Eimeria spp. available in GenBank, the results showed that the lengths of Eimeria intestinalis, Eimeria flavescens and Eimeria magna were 1065, 1009 and 1047 bp, respectively, and the sequence homologies with the same species sequences were 99.2%, 99.0% and 94.5%, respectively, while were 55.3% to 82.1% compared with corresponding sequences of other different species sequences. The phylogenetic analysis using software Mega 5.0 showed that all rabbit coccidia clustered together in a clade, which was divided into two sister lineages, corresponding to the presence or absence of oocyst residuum. The result demonstrated ITS could be used as a molecular marker for the species identification of rabbit coccidia.     

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.     

绵羊球虫感染情况和种类调查

应用饱和蔗糖溶液漂浮法对河南、山东和东北等地的1052只绵羊球虫感染情况及种类进行了调查,结果表明球虫总感染率为94.8%,对968份阳性样本中的球虫卵囊进行形态学鉴定,共检出12种艾美耳球虫,分别为阿撒他艾美耳球虫、巴库艾美耳球虫、小型艾美耳球虫、贡氏艾美耳球虫、类绵羊艾美耳球虫、颗粒艾美耳球虫、苍白艾美耳球虫、马耳西卡艾美耳球虫、温布里吉艾美耳球虫、错乱艾美耳球虫、槌形艾美耳球虫和浮氏艾美耳球虫。调查结果发现绵羊最多可同时感染9种球虫,多数为2~5种,混合感染率为71.8%;1岁以下和1岁以上绵羊球虫感染率分别为99.4%和86.0%,平均OPG值分别为7907.36和3263.89;舍饲和放牧绵羊球虫感染率分别为97.0%和89.0%;夏、秋季为球虫主要流行季节。     

鸡柔嫩艾美耳球虫广西株的分离与鉴定

目的建立一种简单、实用的单卵囊分离方法，并对一株广西柔嫩艾美耳球虫进行分离。方法采用电泳制胶槽来制作琼脂块进行球虫单卵囊的分离，单卵囊实验感染9只1日龄雏鸡，感染后收集粪便，用饱和盐水漂浮法进行卵囊检测。纯种卵囊经口感染10只1日龄雏鸡，观测卵囊寄生部位、最短孢子化时间，以及其潜在期和排卵高峰期。结果2只雏鸡粪便中检出卵囊，单卵囊感染成功率为22％。通过对其中一株球虫的研究，根据其卵囊的形状、大小、寄生部位、潜在期、卵囊最短孢子化时间、排卵高峰期等生物特征，鉴定该株球虫为柔嫩艾美耳球虫（Eimeria tenella）。结论本研究成功建立一种单卵囊分离技术，可作为球虫单卵囊分离的常规方法。     

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.     

湘黄鸡球虫病病原学调查与鉴定

鸡球虫病是危害养鸡业的主要疾病之一,为了解湘黄鸡球虫病病原学流行情况,选择湘黄鸡饲养较集中的衡东、衡山、衡南、衡阳县以及衡阳市郊区五个县区,以地面散养的湘黄鸡为病原学调查对象。通过粪样分肠段采集,采用琼脂薄板单卵囊的分离培养和球虫单卵囊感染试验。经鉴定,结果发现湘黄鸡球虫病的病原体有堆型艾美耳球虫、早熟艾美耳球虫、柔嫩艾美耳球虫、毒害艾美耳球虫、巨型艾美耳球虫等五种球虫。鉴定中,采用4%琼脂糖块将单卵囊分离最合适,此法与其他分离方法比较,简单易行,不需要专门的分离仪器。     

Efficacy of ionophorous anticoccidial drugs against coccidia in farm-reared pheasants (Phasianus colchicus) from Illinois

Litter samples obtained from a ring-necked pheasant propagation farm in Illinois contained coccidia: 57.5% of the oocysts were Eimeria duodenalis, 24.9% were E. tetartooimia, 8.8% were E. phasiani, and 8.8% were E. pacifica. Ionophorous anticoccidial drugs were tested for efficacy against the pheasant coccidia. All three drugs reduced oocyst production and prevented mortality in young pheasants; unmedicated infected controls had a 40% mortality rate. Monensin at 120 ppm in the feed was coccidiocidal against E. duodenalis and E. tetartooimia, partly coccidiocidal against E. pacifica, and only partly coccidiostatic against E. phasiani. Salinomycin at 60 ppm in the feed was highly efficacious and coccidiocidal against all four species, but the salinomycin-medicated pheasants gained the least of all medicated birds. Lasalocid at 120 ppm in the feed was the most effective, with nearly complete coccidiocidal activity against all four coccidial species.     

The coccidia of sheep and goats in Senegal

A survey of the coccidia in domestic sheep and goats was undertaken to ascertain the type of Eimeria species and the number of different coccidial species in individual faecal samples. Simultaneously the prevalence and the oocyst output was investigated in 2234 sheep and 577 goats during a 12-month period. Eight Eimeria species were encountered in sheep: E. ahsata, E. crandallis, E. faurei, E. intricata, E. ovina, E. ovinoidalis, E. pallida and E. parva.

In goats the following species were found: E. ahsata, E. arloingi, E. christenseni, E. crandallis, E. faurei, E. intricata, E. ninakohlyakimovae and E. parva.

The prevalence in sheep was 94% and in goats 85%, multiple parasitism was the rule. No seasonal fluctuation was observed in the prevalence or oocyst output. The sheep and goats' oocyst output was moderate, the mean for both being in the range of 1000–5000 oocysts/g of faeces.