Detection of T. gondii in tissues of sheep and cattle following oral infection.

It has been reported in the literature that cattle are more resistant to toxoplasmosis than sheep. Congenital disease due to T. gondii infection is rarely reported in cattle whereas the parasite is a major cause of abortion and neonatal mortality in sheep. It is believed that sheep remain chronically infected for life. Undercooked meat from infected sheep is an important source of infection for man. In contrast cattle are thought to harbour fewer parasite tissue cysts which may not persist for the lifetime of the host. Therefore, cattle are believed to pose less of a risk for human infection. In this study we examined the presence of T. gondii within a range of tissues in sheep and cattle at 6 weeks and 6 months following oral infection with 10(3) or 10(5) sporulated oocysts of T. gondii. The presence of parasite was determined by bioassay in mice and using polymerase chain reaction (PCR). The results from this study show that T. gondii was more frequently and consistently detected in sheep, in particular within brain and heart tissues, whereas parasites were not detected in the samples of tissues taken from cattle. T. gondii was more frequently detected in sheep given the higher dose of T. gondii. Examination of tissues at either 6 weeks or 6 months after infection did not appear to affect the distribution of T. gondii. The polymerase chain reaction has more specificity and sensitivity when detecting the presence of T. gondii in large animals than histological detection.     

Nested PCR-based detection of Toxoplasma gondii in German shepherd dogs and stray cats in South Korea

The prevalence of Toxoplasma gondii was surveyed by using a nested polymerase chain reaction (PCR) that was targeted to T. gondii B1 gene in German shepherd dogs and stray cats. Sixty-four (46.3%) out of 138 German shepherd dogs and 50 (47.2%) out of 106 stray cats were tested positive by the nested PCR assay, respectively. There was no significant difference in gender or age in German shepherd dogs and stray cats. In the five positive dogs and five positive cats, the nucleotide partial sequence of the T. gondii B1 gene was identified by direct sequence analysis. All the sequences were identical to each other and the corresponding sequence, T. gondii B1 gene (Accession No. AF179871). The results suggest that the prevalence of T. gondii is high, and the nested PCR assay is useful for early detection of T. gondii for asymptomatic dogs and cats.     

Comparison of different commercial DNA extraction kits to detect Toxoplasma gondii oocysts in cat faeces

The cat is the definitive host of Toxoplasma gondii and plays an important role in the transmission of this and other coccidian parasites, e.g. Hammondia hammondi, a protozoon closely related and morphologically similar to T. gondii. A number of techniques to detect T. gondii nucleic acids in feline faeces are described and several extraction kits for isolating pathogen DNA from faeces or soil are commercially available. To compare the performance of such kits with regard to isolating oocyst DNA, a feline sample that had tested negative for coccidian parasites including T. gondii and H. hammondi was spiked with 10(4), 10(3), 10(2), 50 and 10 H. hammondi oocysts. Several ready-to-use stool or soil kits and an in-house method were then used to extract parasite DNA from these spiked faecal samples. Of six kits tested, two were found suitable for the detection of H. hammondi oocysts DNA by the polymerase chain reaction (PCR) in faecal samples with a detection limit of 250 oocysts per 1 g of faecal sample. These two kits revealed a similar, even slightly lower detection limit (50 oocysts per 1 g of sample) when tested with faecal samples spiked with T gondii oocysts.     

Use of a multiplex polymerase chain reaction assay in the antemortem diagnosis of toxoplasmosis and neosporosis in the central nervous system of cats and dogs

OBJECTIVE: To develop a multiplex polymerase chain reaction (PCR) assay for the detection of Toxoplasma gondii and Neospora caninum DNA in canine and feline biological samples. SAMPLE POPULATION; Biological samples from 7 cats with systemic (n = 4) or CNS (3) toxoplasmosis, 6 dogs with neospora- or toxoplasma-associated encephalitis, and 11 animals with nonprotozoal disease. PROCEDURE: Primers for T gondii, N caninum, and the canine ferritin gene (dogs) or feline histone 3.3 gene (cats) were combined in a single PCR assay. The DNA was extracted from paraffin-embedded brain tissue, CSF, or skeletal muscle. The PCR products with positive results were cloned, and sequence identity was confirmed. RESULTS: Of 7 cats and 4 dogs with immunohistochemical or serologic evidence of toxoplasmosis, PCR results were positive for all cats and 3 dogs for T gondii, and positive for T gondii and N caninum for 1 dog. Another dog had negative PCR results for both parasites. Of 2 dogs with immunohistochemical or serologic evidence of neosporosis, PCR results were positive for 1 for N caninum and positive for the other for T gondii. All negative-control samples yielded negative results for T gondii and N caninum on the PCR assay. CONCLUSIONS AND CLINICAL RELEVANCE: Standard tests for toxoplasmosis or neosporosis associated with the CNS rely on serologic, histologic, or immunohistochemical analysis and can be difficult to interpret. The multiplex PCR assay with built-in control reactions could be a complementary clinical tool for the antemortem diagnosis of toxoplasmosis or neosporosis associated with the CNS.     

Single tube nested PCR for the detection of Toxoplasma gondii in fetal tissues from naturally aborted ewes.

A single tube nested polymerase chain reaction (PCR) assay targeting the multicopy 18S-5.8S rRNA internal transcribed spacer (ITS1) region has been developed for the diagnosis of Toxoplasma gondii-induced abortion in ovine fetal tissues. In all, 145 ovine fetal samples including brain, spleen, lung, liver, kidney, placenta and fetal fluids from 53 fetuses and stillborns of 32 farms in Northern Spain were analyzed. Thirty-six samples belonging to nine fetuses and one stillborn lamb were T. gondii PCR-positive. Although T. gondii DNA was amplified from different types of tissues, brain was the tissue with the highest detection rate. All animals that had histopathological lesions associated to T. gondii infection were positive by PCR. In addition, four fetuses whose histological examination was hindered by autolysis were PCR-positive. Results obtained by PCR and indirect fluorescent antibody test (IFAT) showed good correspondence, demonstrating the diagnostic value of the two techniques. However, PCR has the advantage over serology in its ability to diagnose T. gondii infection at earlier stages of gestation when the fetus is not yet immunocompetent and in lambs that have taken colostrum. Once other abortifacient agents are ruled out, PCR detection of the ITS1 region in fetal tissues is a valuable and relatively rapid technique for the diagnosis of ovine abortion caused by T. gondii.     

Comparison of detection methods for Toxoplasma gondii in naturally and experimentally infected swine

Results from recent serological surveys and epidemiological studies show that pigs raised in a variety of management systems can be carriers of the tissue cyst stage of Toxoplasma gondi. This parasite can be transmitted to humans through the consumption of improperly prepared pork, making detection and removal of infected swine carcasses from the food chain an important food safety issue. Several methods are available for detection of T. gondii infected swine, including serological assays, polymerase chain reaction, and animal bioassays. The aim of the present study was to compare the detection sensitivities of six of these commonly used methods for detection of T. gondii infection in tissues from naturally and experimentally infected pigs. The results indicate that a serum-based ELISA is the most sensitive method, of those tested, for detection of T. gondii infected swine.     

3种实验动物相关病原液相芯片检测方法的建立

为建立快速高通量检测实验动物质量相关布鲁菌、沙门菌、弓形虫3种病原体的方法,根据其序列设计引物及探针,探针经修饰后与荧光编码微球偶联,将偶联后的探针与PCR产物杂交反应,通过液相芯片检测仪(Luminex200)检测荧光信号,分析实验动物感染布鲁菌、沙门菌和弓形虫的情况.结果显示:初步建立了可同时检测布鲁菌、沙门菌和弓...     

猪弓形虫特异性PCR诊断方法的建立及应用

以猪弓形虫核糖体DNA第一内转录间隔区(ITS1)序列为模板自行设计引物,建立了猪弓形虫病特异PCR诊断方法,并从弓形虫国际标准强毒株RH速殖子和疑似T.gondii感染猪全血及肺脏组织样品基因组DNA中扩增出了预期长度273bp的目的DNA片段。敏感性和特异性试验结果显示,该PCR方法能检测到的最低DNA量为0.001ng,且与相关的9种对照寄生虫、细菌和病毒无交叉反应。用建立的PCR诊断方法对临床30份猪弓形虫疑似病料和60份健康猪抗凝全血样品进行检测,结果30份病料中有24份呈现阳性;60份健康猪血中有5份为阳性;随机取两个临床样品的阳性PCR扩增片段进行克隆测序表明,二者序列与Gen-Bank中已登录的猪弓形虫ITS1基因相应部分序列完全相同。以上表明所建立的PCR方法具有高度的敏感性和特异性;本研究为猪弓形虫病的快速诊断提供了一种新方法。     

Comparison of loop-mediated isothermal amplification (LAMP) and real-time PCR method targeting a 529-bp repeat element for diagnosis of toxoplasmosis

Loop-mediated isothermal amplification (LAMP) is a simple method that can amplify DNA with high specificity, sensitivity, and rapidity. In this study, we compared the performance of LAMP and real-time PCR assays for diagnosis of toxoplasmosis. We designed a real-time PCR assay targeting a 529 bp element repeated 200-300 times in the Toxoplasma gondii genome. The detection limits of the LAMP and real-time PCR assays were 10 fg/μL and 1 fg/μL of T. gondii DNA, respectively. Conventional PCR, LAMP, and real-time PCR methods were applied to detect T. gondii DNA in blood samples from 284 pigs and 292 sheep. Positive results were obtained with 0.4%, 3.2%, and 4.2% of the pig samples and 3.8%, 17.1%, and 17.8% of the sheep samples with conventional PCR, LAMP, and real-time PCR analyses, respectively. The real-time PCR assay provided the most sensitive diagnosis of toxoplasmosis, but the LAMP assay has potential as an alternative tool for detection of T. gondii in the field.     

弓形虫棒状蛋白17基因的克隆与序列分析

为研究弓形虫棒状体蛋白17(ROP17)基因的遗传变异,本研究以弓形虫RH株、PRU株和TgC7株为研究对象,首次PCR扩增了其ROP17基因部分序列,将PCR产物纯化后克隆到pMD18-T并测序。将测定的序列与网上下载的弓形虫VEG株、GT1株和ME49株相应序列进行比对,然后用Mega 5.0程序的NJ法和Puzzle 5.2程序的ML法构建系统发育树。结果表明,3株弓形虫分离株的ROP17基因部分序列长度均为1375 bp,A+T含量在49.53%~50.04%之间,3个虫株相应序列的变异碱基数皆小于20个,其变异率为2.3%,氨基酸序列变异率在0~6.11%之间。系统发育结果显示, ROP17基因部分序列能区分弓形虫基因Ⅰ型和Ⅱ型的虫株。本研究结果为进一步研究弓形虫ROP17基因的变异及研制弓形虫ROP17基因的亚单位疫苗提供了理论依据。     

Detection of Toxoplasma gondii in the milk of experimentally infected lactating cats.

Tachyzoites of Toxoplasma gondii have been found in the milk of sheep, goats, cows and mice and infection by ingestion of raw goat milk has been documented in humans. Lactational transmission from infected cats to their kittens is suspected but the organism has not been detected in the milk. The purpose of this study was to demonstrate the presence of T. gondii in the milk of experimentally infected cats. Pregnant specific pathogen free cats were inoculated orally with T. gondii at various times prior to parturition. Feces were examined for oocyst shedding after sugar solution centrifugation. Milk was collected for polymerase chain reaction (PCR) and bioassay in mice. T. gondii was detected in the milk of five of six cats by either bioassay or PCR.     

牛瑟氏泰勒虫ITS基因PCR诊断方法的建立

本试验根据GenBank上登录的牛瑟氏泰勒虫ITS基因序列(AY661522.1),应用Primer Premier 5.0和Oligo 6.31软件设计合成1对特异性引物,以牛瑟氏泰勒虫DNA为模板,建立了牛瑟氏泰勒虫ITS基因PCR诊断方法。该方法扩增片段大小为1020 bp,与参考序列的同源性为98%;建立的PCR方法与猪附红细胞体、犬新孢子虫和弓形虫均无交叉反应,最低DNA检出量为1.5 pg/μL;通过对60份临床样品的检测,并与血涂片方法进行比较,结果显示PCR方法具有特异、敏感等特点,适用于牛瑟氏泰勒虫的检测。     

The detection of Toxoplasma gondii by comparing cytology, histopathology, bioassay in mice, and the polymerase chain reaction (PCR)

The objective of this study was to compare the different methods of detecting Toxoplasma gondii in sheep tissue, tested serologically positive by the indirect immunofluorescent antibody test (IFAT). Brain, diaphragm, and blood samples were collected from 522 sheep slaughtered at the S?o Manuel abattoir, S?o Paulo State, Brazil. Brain and diaphragm samples from IFAT seropositive animals were digested by both trypsin and pepsin and then injected into mice. Part of the digested samples was used to prepare slides for Giemsa staining and in the polymerase chain reaction (PCR). Tissue fragments were fixed in formalin and examined using hematoxilin-eosin (HE). Forty of the sheep (7.7%) were IFAT positive. T. gondii was isolated in 23 (59.0%) of the 39 mice with pepsin-digested brain samples and in 27 (69.0%) of the 39 with trypsin-digested brain samples. Injection of diaphragm samples led to T. gondii isolation in 26 (66.7%) of the 39 pepsin-digested samples and 21 (53.8%) of the 39 trypsin-digested samples. Cytological and hystopathological examination of both brains and diaphragms was negative in all examined sheep. PCR was positive in 7 (17.9%) of the trypsin and 2 (5.1%) of the pepsin-digested samples, while 9 (23.1%) of the trypsin and 3 (7.7%) of the pepsin-digested samples showed T. gondii DNA. T. gondii isolation rate in mice (n = 34; 85.0%) was significantly higher than detection by PCR (n = 15; 37.5%).     

牛卵形巴贝斯虫巢式PCR诊断方法的建立及初步应用

根据GenBank上发表的牛卵形巴贝斯虫CCTη基因序列设计合成2对巢式PCR引物,建立牛卵形巴贝斯虫巢式PCR诊断方法,对该方法的最佳反应条件进行了筛选,并进行了特异性、敏感性及临床样本检测试验。结果表明,建立的巢式PCR方法外引物扩增牛卵形巴贝斯虫基因组片段的长度为1 008bp,内引物为537bp;该方法扩增不出牛瑟氏泰勒虫、弓形虫、犬新孢子虫基因组DNA;最低检测DNA含量为16fg;通过对46份临床样本的检测,该巢式PCR较常规PCR阳性检出率高8.7%。本试验为牛卵形巴贝斯虫病的诊断提供了一种更为特异、敏感的检测技术。     

3株柔嫩艾美耳球虫28SrRNA基因部分序列的扩增与分析

本研究应用PCR技术扩增来自广东的3株柔嫩艾美耳球虫的28S rRNA基因部分序列,并与GenBank^TM登录的柔嫩艾美耳球虫、堆型艾美耳球虫、鼠肉孢子虫和刚地弓形虫虫株的相应序列进行比对分析。试验结果显示,柔嫩艾美耳球虫3个样品均获得1172 bp的28S rRNA基因部分有效序列,不同虫株序列没有差异,与GenBank^TM登录的柔嫩艾美耳球虫相应序列只有一个碱基差异,显示种内序列高度保守,而与堆型艾美耳球虫、鼠肉孢子虫、刚地弓形虫相应的序列存在不同程度的差异。结果表明,28S rRNA基因部分序列可作为研究艾美耳球虫种间及其他顶复门原虫遗传变异的标记。     

刚地弓形虫18S rDNA基因的克隆与序列分析

采用PCR方法从弓形虫RH株和CN株总DNA中分别扩增到18S rDNA基因，与pGEM-T easy vector连接，并进行DNA测序分析。结果表明，2个虫株均扩增出1745bp的片段，用DNAMAN软件对其与GenBank上2个虫株相应序列进行同源性比较，4个虫株的同源性为99．34％。刚地弓形虫虫株在不同宿主和不同区域上存在着一定的差异。     

检测实验动物弓形虫感染的两种PCR方法的建立和比较

为了建立敏感、稳定、特异的PCR检测体系，用于实验动物弓形虫感染的检测。采用B1基因设计引物，建立常规PCR，用P30基因设计引物，建立巢式PCR；用两种PCR方法检测实验感染弓形虫小鼠血液和腹腔波中的DNA动态变化；用巢式PCR检测自然状态下的普通级豚鼠、教学和科研用兔的弓形虫感染率，并和常规PCR检到结果比较。结果，巢式PCR可检测到1fgDNA含量，比常规PCR敏感lOO倍；对其他微生物DNA无交叉现象，特异性强；对同一样品重复检测3次，阴、阳性结果一致，稳定性好。小鼠感染弓形虫2d后，巢式PCR对小民腹腔液的阳性检出率为83．3％，对血液的阳性检出率为33．3％；感染3d后，腹腔液阳性检出率为100％；而常规PCR在小鼠感染3d和4d后才能在腹腔液和血液中检测到，检出率各为16．7％。受检普通级豚鼠没有感染弓形虫，教学和科研用兔的弓形虫总感染率为14．3％。结论认为，巢式PCR方法可用于实验动物弓形虫早期感染的检测，具有敏感性高、特务性强、稳定性好的特点。     

犬附红细胞体PCR检测方法的建立

目的 步建立犬附红细胞体PCR检测方法。方法 据已发表的附红细胞体基因序列,设计了一对特异性引物,以犬附红细胞体基因组DNA和附红细胞体可疑病犬样品DNA为模板,聚合酶链式反应（PCR）扩增,扩增产物经克隆测序分析。结果 CR扩增产物大小为541bp,序列分析表明与GenBank数据中发表的序列一致,表明这套引物成功扩增出目的基因序列,但正常犬血样品DNA和弓形虫、伊氏锥虫、吉氏巴贝斯虫、犬细小病毒、犬瘟热的DNA样品都不能扩增出目的基因片段。结论 研究建立的PCR方法可以用于犬附红细胞体的检测,为犬附红细胞体病的诊断及分子流行病学的调查提供了新的手段。     

Aetiology of bovine abortion in Switzerland from 1986 to 1995--a retrospective study with emphasis on detection of Neospora caninum and Toxoplasma gondii by PCR

In a retrospective study, covering the period from 1986 to 1995, tissues of aborted fetuses were re-examined. A total of 347 cases were tested immunohistochemically, among them samples of 223 brains were examined for Neospora caninum, Toxoplasma gondii and Bovine Virus Diarrhoea Virus (BVDV), and 249 placentae for Chlamydiaceae. Two real-time PCR assays, one for N. caninum, and one for T. gondii, were developed. These potential abortion-inducing agents were detected - and confirmed by PCR, except for BVDV - in 16.1% (N. caninum), 0% (T. gondii), 9.9% (BVDV) and 0.8% (Chlamydiales) of the cases examined. Immunohistochemistry proved to be inadequate for the detection of the protozoal epitopes, whereas it was confirmed as a very useful tool for the detection of BVDV. In abortion material, PCR is considered to be more suitable for the detection of protozoa and Chlamydophila abortus, an adequate sampling presupposed.     

Occurrence of Toxoplasma gondii and Hammondia hammondi oocysts in the faeces of cats from Germany and other European countries

Faecal samples of 24,106 cats from Germany and other European countries were examined microscopically in a veterinary laboratory in Germany between October 2004 and November 2006 to estimate the prevalence of animals shedding Toxoplasma gondii or Hammondia hammondi oocysts. Oocysts of 9-15 microm size with a morphology similar to that of H. hammondi and T. gondii were found in 74 samples (0.31%). A total of 54 samples were further characterised to achieve a species diagnosis and to determine the genotype of T. gondii isolates by PCR and PCR-RFLP. From these samples, 48 isolates were obtained: 26 (0.11%) were finally identified as T. gondii and 22 (0.09%) as H. hammondi. T. gondii-positive samples came from Germany, Austria, France and Switzerland while H. hammondi was detected in samples from Germany, Austria and Italy. In two samples (one T. gondii and one H. hammondi), PCR indicated the presence of Hammondia heydorni DNA. No Neospora caninum DNA was detected in any of the feline faecal samples. Twenty-two of the 26 T. gondii isolates could be genotyped. A PCR-RFLP analysis for the SAG2, SAG3, GRA6 and BTUB genes revealed T. gondii genotype II in all cases. Morphologically, H. hammondi oocysts exhibited a statistically significantly smaller Length-Width-Ratio than T. gondii oocysts.