牛源隐孢子虫新疆南疆分离株巢式PCR鉴定

为了解新疆南疆牛隐孢子虫病的感染情况,从而为该病的防控提供理论依据,以新疆南疆某牛场牛粪中的隐孢子虫卵囊DNA为模板,根据隐孢子虫18S rRNA序列设计引物,采用巢式PCR方法鉴定了自然感染的新疆南疆牛源隐孢子虫,并对扩增出的目的片段进行了测序、同源性分析,并运用MEGA 5.0软件构建系统发育进化树。结果表明:检测材料能够扩增出目的片段;新疆南疆牛源隐孢子虫分离株的18S rRNA与广州微小隐孢子虫的同源性达100%,种系发育亲缘关系最近,处于同一分支,与宁夏、河南、黑龙江等地微小隐孢子虫的同源性达99%。说明新疆南疆牛源隐孢子虫分离株在种属上为微小隐孢子虫。     

陕西部分地区秦川牛断奶后犊牛隐孢子虫感染状况

为明确陕西部分地区秦川牛断奶后犊牛隐孢子虫感染率及其种类分布,本试验从杨凌和宝鸡3个秦川牛场采集了113份3~6月龄的犊牛粪便样品,基于隐孢子虫18SrRNA基因位点对其进行PCR检测和序列分析。结果显示,隐孢子虫总感染率为16.8%(19/113),各养殖场的隐孢子虫感染率差异不显著(P0.05);种类鉴定发现了3种隐孢子虫,分别为安氏隐孢子虫(Cryptosporidium andersoni)、瑞氏隐孢子虫(Cryptosporidium ryanae)和牛隐孢子虫(Cryptosporidium bovis),其中安氏隐孢子虫为优势虫种,广泛分布于各个养殖场。这些研究结果表明这些地区的秦川牛断奶后犊牛隐孢子虫感染比较普遍,且种类较为复杂。     

玉溪霍尔多巴吉鹅感染贝氏隐孢子虫的分子鉴定

为了对云南省玉溪市某养殖场的霍尔多巴吉鹅进行隐孢子虫检测,本试验采集疑似感染隐孢子虫的霍尔多巴吉鹅的粪便样品,利用饱和盐水漂浮法进行镜检,并结合分子生物学鉴定方法,提取粪便样品DNA,采用巢氏PCR扩增隐孢子虫核糖体小亚基RNA(SSU rRNA)基因,测序后通过NCBI进行Blast在线比对,构建系统发育进化树。结果显示,所采集的鹅粪便样品中,隐孢子虫检出率为20.45%(9/44);隐孢子虫SSU rRNA基因的PCR扩增结果显示,该鹅源隐孢子虫与贝氏隐孢子虫同源性达100%;系统进化分析显示,二者位于同一分支。结果表明,该养殖场的霍尔多巴吉鹅存在贝氏隐孢子虫感染。     

柯坪县规模化养殖场双峰驼隐孢子虫感染情况调查与种类鉴定

[目的]掌握新疆维吾尔自治区阿克苏地区柯坪县规模化养殖场双峰驼隐孢子虫的感染情况和种类分布情况。[方法]从柯坪县4个乡(镇)6个规模化双峰驼养殖场共收集516份粪便样本,全部提取DNA样本,基于隐孢子虫(Cryptosporidium)SSU rDNA序列进行PCR检测,序列比对分析后进行隐孢子虫种类鉴定;采用χ²检验法比较不同规模化养殖场双峰驼隐孢子虫的感染率差异;将被鉴定为微小隐孢子虫(Cryptosporidium parvum,C. parvum)阳性的DNA样本,基于gp60基因序列进行PCR检测,序列比对分析后进行微小隐孢子虫亚型鉴定。[结果]516份双峰驼粪便DNA样本中检测出34份隐孢子虫阳性,总体感染率为6.59%(34/516),以阿恰勒镇养殖场2的双峰驼隐孢子虫感染率最高,为9.57%(9/94);不同养殖场双峰驼隐孢子虫的感染率统计学差异显著(χ²=5.497,P<0.05)。基于SSU rDNA序列,34条隐孢子虫序列经比对分析,鉴定出3种隐孢子虫,分别为安氏隐孢子虫(n=29)、隐孢子虫大鼠基因型Ⅳ(n=1)...     

新疆南疆某规模化绵羊养殖场腹泻羔羊隐孢子虫感染情况及其分子鉴定

[目的]了解新疆南疆某规模化绵羊养殖场腹泻羔羊隐孢子虫感染情况和基因亚型分布特点。[方法]采集新疆维吾尔自治区某规模化绵羊养殖场4个品种1月龄以内腹泻羔羊新鲜粪便样本60份,使用饱和蔗糖溶液漂浮法检查隐孢子虫卵囊,进行种类初步鉴定;全部粪便样本提取基因组DNA后,基于隐孢子虫SSU rRNA基因位点和微小隐孢子虫gp60基因位点,对其进行PCR扩增、测序和序列分析,鉴定隐孢子虫种属和基因亚型,构建遗传进化树解析其分子遗传特征。[结果]经显微镜观察,发现38份样本呈隐孢子虫卵囊阳性,形态学初步鉴定为微小隐孢子虫;基于SSU rRNA基因位点,采用PCR方法检测出52份样本呈隐孢子虫阳性,感染率为86.67%(52/60),经序列比对分析,均为微小隐孢子虫;基于微小隐孢子虫gp60基因位点,PCR扩增后成功获得49条序列,经比对分析均为ⅡdA19G1基因亚型。[结论]该养殖场腹泻羔羊普遍感染微小隐孢子虫,其基因亚型均为ⅡdA19G1。调查结果为新疆南疆绵羊隐孢子虫种属鉴定与遗传进化研究提供了基础数据。     

印度西孟加拉奶牛隐孢子虫人畜共患传播的分子特性及评估

过去很少开展印度奶牛隐孢子虫遗传多样性和在人畜共患潜力方面的研究。为了评估奶牛作为人感染隐孢子虫传染源的重要性,从印度西孟加拉2个奶牛场采集180头奶牛和50位养殖工人的粪样,用PCR方法扩增隐孢子虫DNA序列,通过PCR-RFLP分析进行隐孢子虫18S rRNA基因分型。对本试验扩增的DNA序列和GenBank公布的相关序列进行系统发育分析。青年奶牛的隐孢子虫感染率高于成年奶牛的感染率,总感染率为11.7%。微细隐孢子虫、牛隐孢子虫等在牛上的发生率呈年龄相关。在1份小牛样品中检测到了猪隐孢子虫基因型DNA。养殖场工人感染了人隐孢子虫、微细隐孢子虫和新型牛隐孢子虫。试验结果表明,印度奶牛场存在人和奶牛之间传播隐孢子虫的潜在风险。     

江苏部分集约化养殖场肉羊隐孢子虫分子流行病学调查及其虫种鉴定

研究旨在了解江苏部分集约化养殖场肉羊隐孢子虫的流行情况,采集江苏省8个集约化养殖场的446份肉羊粪便样品。根据隐孢子虫的基因序列,运用巢式PCR方法,检测肉羊粪便样品的隐孢子虫感染情况,再通过测序等方法鉴定其虫种类型。结果显示：被检样品中肉羊隐孢子虫的总感染率为4.48%(20/446),经鉴定有2种虫株,分别为泛在隐孢子虫和微小隐孢子虫。研究表明,养殖场内肉羊存在隐孢子虫的感染,可能诱发其他传染病和人畜共患病等问题。     

新疆部分地区褐牛肠道寄生虫感染情况调查

为了解新疆褐牛肠道寄生虫感染情况,试验采用饱和蔗糖溶液漂浮法对采自新疆9个地区的524份褐牛新鲜粪便样品进行检查,并对检查结果按不同地区、年龄、饲养模式下褐牛肠道寄生虫感染率的差异进行分析。结果表明:211份样品肠道寄生虫阳性,总感染率为40. 3%;检出7种寄生虫,以球虫为优势感染虫种,感染率为32. 8%;隐孢子虫、圆线虫、细颈线虫、鞭虫、莫尼茨绦虫和双腔吸虫感染率分别为2. 5%、9. 5%、0. 8%、1. 0%、1. 5%和1. 0%,混合感染率为7. 3%。不同饲养模式下(圈养、散养)及不同季节(夏季和冬季)间褐牛肠道寄生虫感染率差异显著(P0. 05),不同年龄段间差异不显著(P0. 05)。说明新疆地区褐牛肠道寄生虫感染较普遍,应加强其防控工作。     

不同养殖模式下山羊隐孢子虫感染分析及基因型鉴定

为探讨四川部分地区不同养殖模式下成年山羊隐孢子虫的感染差异以及感染虫种基因型,分别采集了规模化养殖模式和散养模式各6个养殖场共342份新鲜粪便,采用改良饱和蔗糖溶液漂浮法处理后提取粪便DNA,经套式PCR扩增18S rRNA基因,产物测序后进行遗传进化分析。结果:采样地区山羊隐孢子虫总感染率为4.7%,12个养殖场中4个养殖场(名山、纳溪、邻水和双流)存在隐孢子虫感染,感染率2.5%～19.2%,且4个隐孢子虫感染阳性场均为散养模式。序列分析表明,感染虫种为肖氏隐孢子虫(Cryptosporidium xiaoi)和猪隐孢子虫(C.suis),并以肖氏隐孢子虫为主(68.7%)。本试验初步表明,规模化养殖模式和散养模式下山羊隐孢子虫感染存在差异(P0.01);首次在成年山羊中检出猪隐孢子虫虫种,序列分析表明所获得的猪隐孢子虫序列与人源猪隐孢子虫序列完全一致,提示此次分离的羊源猪隐孢子虫为人兽共患隐孢子虫种。     

应用PCR检测隐孢子虫卵囊的研究

隐孢子虫病是一种重要的人畜共患原虫病。为了在临床样品中更准确、快速地检测隐孢子虫卵囊，从初步纯化的含有不同数量隐孢子虫卵囊的样品中和含有不同数量隐孢子虫卵囊的奶牛粪便中，直接提取DNA或用DNA纯化试剂盒对提取的奶牛粪便中卵囊DNA进行纯化之后用作PCR模板，用1对人工合成寡核苷酸作为PCR引物，扩增片段大小为452bp。优化了Mg^2 浓度、引物浓度和dNTP浓度，并进行了特异性检验。建立的PCR具有隐孢子虫属特异性，不仅扩增出新鲜样品DNA提取物中的目的片段，而且扩增出放置6年之久的DNA提取物中的目的片段。样品经过初步纯化之后，最低检测值100个卵囊／ml；从含有隐孢子虫卵囊的奶牛粪便中提取DNA，尔后经过DNA纯化试剂盒纯化，PCR最低检测值为10^5个卵囊/g粪便。     

新疆褐牛与美新褐牛生长性能比较分析研究

[目的]旨在为今后新疆褐牛品质选育提高工作奠定基础.[方法]通过对伊犁新褐种牛场和伊犁尼勒克县牧强新疆褐牛种畜场的牛群进行体型外貌鉴定,测定1～10岁纯种新疆褐牛和美国瑞士褐牛冻精冷配改良后代母牛关新F1、F2代的生长性能指标,对数据进行统计分析.[结果]显示,美新F1、F2代不同生长阶段各生长性能指标均显著（P＜0.05）或P＜0.05的高于纯种新疆褐牛.[结论]美国瑞士褐牛优良基因对提高新疆褐牛体尺和体重等指标效果显著.     

Sample prevalence and molecular characterisation of Cryptosporidium andersoni within a dairy herd in the United Kingdom

Bovine cryptosporidiosis is usually an acute diarrhoeal disease of young calves caused by Cryptosporidium parvum. However, chronic infection with Cryptosporidium andersoni has been associated with gastritis, reduced milk yield and poor weight gain in adult cattle. Here we describe the first genetic confirmation and characterisation of C. andersoni from cattle in the United Kingdom and its sample prevalence within a dairy herd. Oocysts measured 7.5+/-0.4 microm x 5.5+/-0.4 microm (7.0-8.5 microm x 4.5-6.5 microm) with a length-to-width ratio of 1.37 (1.08-1.60). The within-herd sample prevalence was 16% (95% confidence intervals=10.4-21.6%). Nested polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) and sequence analysis of the small subunit rDNA was used to confirm the species and characterise the isolates. Due to the lack of overt, acute, clinical symptoms, the incidence, prevalence and importance of this parasite is probably currently underestimated in cattle in the UK. The potential for zoonotic transmission is unknown.     

Molecular prevalence of Cryptosporidium spp. in dairy calves in Southern states of India

Dung samples were collected from dairy calves of south Indian states viz., Andhra Pradesh, Karnataka, Kerala, Tamil Nadu and union territory, Puducherry and are subjected to nested polymerase chain reaction (PCR) targeting 18S rRNA gene for detection of Cryptosporidium infection. Of the 459 dung samples screened 182 were found positive with a prevalence of 39.65%. Highest prevalence of Cryptosporidium was observed in Puducherry (86.67%) and lowest in Kerala (17.65%). Genotyping by PCR-restriction fragment length polymorphism (RFLP) and sequence analysis revealed the presence of all the four major Cryptosporidium species of cattle viz., Cryptosporidium andersoni, Cryptosporidium ryanae, Cryptosporidium parvum and Cryptosporidium bovis. C. andersoni was widely distributed in calves of Tamil Nadu, Karnataka and Puducherry whereas in Andhra Pradesh C. ryanae was the major species. Of the 64 samples subjected to PCR-RFLP, 39 (60.94%) could be classified as C. andersoni, 18 (28.13%) as C. ryanae, 4 (6.25%) as C. parvum and 3 (4.69%) were confirmed as C. bovis. The results were also confirmed by sequencing of 19 Cryptosporidium DNA samples.     

The potential for zoonotic transmission of Giardia duodenalis and Cryptosporidium spp. from beef and dairy cattle in Ontario, Canada

The objective of this study was to compare the occurrence and the genotypes and species of Giardia duodenalis and Cryptosporidium spp. in beef and dairy cattle from farms in the Regional Municipality of Waterloo, Ontario, in an effort to determine the potential for zoonotic transmission from these animals. Pooled manure samples were collected from 45 dairy cattle farms and 30 beef cattle farms. The presence of Giardia cysts and Cryptosporidium oocysts was determined by immunofluorescence microscopy, while nested-PCR and DNA sequencing were used to determine genotypes and species. The overall farm prevalence was very high for both Giardia and Cryptosporidium, and was similar for dairy cattle farms (96 and 64%, respectively) and beef cattle farms (97 and 63%, respectively). However, on dairy cattle farms, G. duodenalis and Cryptosporidium spp. were detected in 44% and 6% of total pooled pen manure samples, respectively, with the occurrence of both parasites being generally higher in calves than in older animals. Most Giardia isolates were identified as either the host-adapted genotype G. duodenalis Assemblage E or the zoonotic Assemblage B. Cryptosporidium parvum and Cryptosporidium andersoni were the most frequently identified species in dairy cattle, while the non-zoonotic species Cryptosporidium ryanae and Cryptosporidium bovis were also found. On beef cattle farms, 72% and 27% of the total pooled pen manure samples were positive for Giardia and Cryptosporidium, respectively, with no obvious correlation with age. All Giardia isolates in beef cattle were identified as G. duodenalis Assemblage E, while all Cryptosporidium isolates were identified by sequence analysis as C. andersoni, although microscopic analyses, and subsequent restriction fragment length polymorphism analyses, indicated that other Cryptosporidium species were also present. The results of this study indicate that although Giardia and Cryptosporidium were identified in a higher overall percentage of the pooled beef cattle manure samples than in dairy cattle, firmly established zoonotic genotypes and species were much more common in dairy cattle than in beef cattle in this region. Dairy cattle, and especially dairy calves, may, therefore, pose a greater risk of infection to humans than beef cattle. However, these results may also provide evidence of potential zooanthroponotic transmission (human to animal).     

Cryptosporidium andersoni in Western Australian feedlot cattle

Cryptosporidium andersoni has not been previously reported in feedlot beef cattle in Western Australia. Faecal samples were collected from 10 groups of cattle ranging in age from 11 to 36 months in five different feedlots in Western Australia. The incidence of C. andersoni ranged from 0% to 26%. There were no clinical signs associated with C. andersoni infection, but there was a significant reduction in rate of gain of 0.44 kg in infected animals compared with negative pen mates. Cryptosporidium andersoni is characterised by large oocysts (7.4 x 5.5 μm) and was confirmed by 18S sequencing.     

新疆褐牛0～3月龄犊牛饲养管理技术探讨

新疆褐牛属于乳肉兼用品种,主要分布在新疆伊犁河谷,其具有适应性,抗病力强的特征,适宜农牧区养殖。本文通过从犊牛消化特点来探讨新疆褐牛0~3月龄的饲养管理的关键技术,并结合实际,提出新疆褐牛犊牛早期断奶技术中的注意事项和时间,旨在为广大农牧区新疆褐牛犊牛0~3月龄饲养管理方面提供理论技术指导。     

Cryptosporidium andersoni from a Danish cattle herd: identification and preliminary characterisation

In November 1997, Cryptosporidium andersoni, for the first time, was isolated from a Danish heifer. The isolate was characterised morphologically, molecularly, and furthermore inoculated into mice and one calf. Data on the distribution of cryptosporidia in the herd of origin were obtained at two separate visits in December 1997 and April 1998. C. andersoni was detected in 27 (19.0%) of 142 cattle examined at the first visit, whereas C. parvum was found in six (4.2%). At the following visit 42 (28.0%) of 150 cattle excreted C. andersoni, while 25 (16.7%) were positive for C. parvum. Oocysts of the Danish C. andersoni isolate were ovoid, 7.3(6.5-8.0) x 5.7(5.0-7.0) microm(2) (n=25), with smooth, colourless, single layer oocyst wall and distinct oocyst residuum. The length to width ratio was 1.27 (1.14-1.40, n=25). The identification was verified by sequencing of a 246bp fragment of the rDNA, which was identical to Cryptosporidium muris, the calf genotype (AF093496). The Danish C. andersoni isolate was not transmissible to mice, whereas oocysts were detected in the faeces of one experimentally infected calf from 25 days post-infection (DPI) and shed intermittently at low numbers until 165 DPI, the day of euthanasia. No macroscopic or microscopic changes that could be attributed to infection with C. andersoni were seen in the gastro-intestinal tract of the experimentally infected calf following necropsy and histological examination. This is to our knowledge the first report of C. andersoni in Scandinavia.     

Prevalence of species and genotypes of Cryptosporidium found in 1-2-year-old dairy cattle in the eastern United States

The prevalence of Cryptosporidium species in 1-2-year-old heifers was determined for 571 animals on 14 dairy farms in seven states on the East Coast of the United States. A fecal specimen collected directly from each heifer was processed to concentrate oocysts that were then examined by polymerase chain reaction (PCR). For every PCR-positive specimen the 18S rRNA gene of Cryptosporidium was sequenced. Cryptosporidium was identified by PCR from heifers on 13 of 14 farms. On all except four farms groups of heifers were housed in a barn or in large covered pens. Others were pastured. From many of the same farms an earlier study reported that 41% of 393 pre-weaned calves and 26.2% of 447 post-weaned calves were infected. In the present study, 11.9% of 571 heifers were infected with Cryptosporidium, 0.7% with Cryptosporidium parvum, the zoonotic species. Of 68 PCR-positive specimens characterized by gene sequencing 1, 4, 10, 24, and 29 calves were infected with Cryptosporidium suis, Cryptosporidium parvum, Cryptosporidium deer-like genotype, Cryptosporidium bovis, and Cryptosporidium andersoni, respectively. These findings demonstrate a lower prevalence of infection in 1-2-year-old dairy cattle than in younger cattle as well as a change in the diversity of species present. Consequently, the risk of humans acquiring infection with C. parvum from exposure to feces from yearling and older cattle appears much lower than from exposure to pre-weaned calves.     

Infectivity, pathogenicity, and genetic characteristics of mammalian gastric Cryptosporidium spp. in domestic ruminants.

Farm ruminants were infected experimentally with four mammalian gastric Cryptosporidium, namely Cryptosporidium andersoni LI03 originated from cattle and three isolates of Cryptosporidium muris from brown rat (isolate RN66), Bactrian camel (isolate CB03) and firstly characterized isolate from East African mole rat (isolate TS03). Sequence characterizations of the small-subunit rRNA gene showed that the LI03 isolate was C. andersoni and the other three isolates belonged to C. muris, although the TS03 isolate showed unique sequence variations (one single nucleotide change and four nucleotide insertions). C. andersoni LI03 was infectious for calves only, whereas lambs and kids were susceptible to C. muris CB03. C. muris TS03 and RN66 were not infectious for any farm ruminants. Infection dynamics including prepatent and patent period and infection intensity of the isolates used differed depending on the host species, but no clinical signs of cryptosporidiosis were observed in any of experimentally infected hosts. Cryptosporidium developmental stages were only detected in infected animals in the abomasum region. Histopathological changes were characterized by dilatation and epithelial metaplasia of infected gastric glands with no significant inflammatory responses in the lamina propria.     

Cryptosporidium species detected in calves and cattle in Dagoretti, Nairobi, Kenya

A total of 1,734 cattle faecal samples from 296 dairy-keeping households were collected from urban settings in Nairobi, Kenya. Modified Ziehl-Neelsen staining method and an immunofluorescence assay were used to identify those samples with Cryptosporidium oocyst infection. Oocysts from positive faecal samples were isolated by Sheather's sucrose flotation method and picked from the concentrate using cover slips. Genomic DNA was extracted from 124 of the faecal samples that were positive for Cryptosporidium and was used as template for nested PCR of the 18S rRNA gene. Twenty-five samples (20?%) were PCR-positive for Cryptosporidium, and 24 of the PCR products were successfully cloned and sequenced. Sequence and phylogenetic analysis identified 17 samples (68?%) as Cryptosporidium parvum-like, four samples (16?%) as Cryptosporidium ryanae, three samples (12?%) as Cryptosporidium andersoni and one sample (4?%) as Cryptosporidium hominis. To the best of our knowledge, this is the first genotyping study to report C. parvum-like, C. andersoni and C. hominis in cattle from Kenya. The results of this study show Cryptosporidium infections in calves and cattle may be potential zoonotic reservoirs of the parasite that infects humans.