弓形虫SAG1基因在蜥蜴利什曼原虫中的表达

利用PCR扩增技术从弓形虫RH株全基因组中扩增出弓形虫抗原基因SAG1,将其克隆入真核表达载体质粒pLEXSY-neo2,构建真核表达穿梭质粒pLEXSY-neo2-SAG1。利用电穿孔转染技术将外源基因表达盒转入蜥蜴利什曼原虫,经G418筛选阳性克隆。表达的外源重组蛋白经SDS-PAGE和Western blot进行鉴定,证明SAG1重组蛋白在蜥蜴利什曼原虫中得到表达。     

Seroprevalence of Toxoplasma gondii infection in Norwegian dairy goats

Background

Toxoplasma gondii is a major problem for the sheep industry as it may cause reproduction problems. The importance of T. gondii in Norwegian goat herds is uncertain, but outbreaks of toxoplasmosis in dairy goat farms have been recorded. The aim of this study was to describe the prevalence of T. gondii infection in Norwegian dairy goats by using serology.

Findings

Goat serum originally collected as part of two nationwide surveillance and control programmes between 2002 and 2008 were examined for T. gondii antibodies by using direct agglutination test. In total, 55 of 73 herds (75%) had one or more serologically positive animals, while 377 of 2188 (17%) of the individual samples tested positive for T. gondii antibodies.

Conclusions

This is the first prevalence study of T. gondii infection in Norwegian goats. The results show that Norwegian goat herds are commonly exposed to T. gondii. Nevertheless, the majority of goat herds have a low prevalence of antibody positive animals, which make them vulnerable to infections with T. gondii during the gestation period.     

Porcine abortion outbreak associated with Toxoplasma gondii in Jeju Island, Korea

This report deals with the acute onset of an abortion outbreak and high sow mortality in one pig herd consisted of 1,200 pigs and 120 sows on Jeju Island, Korea. Affected pregnant sows showed clinical signs, including high fever, gradual anorexia, vomiting, depression, recumbency, prostration, abortion, and a few deaths. Four dead sows, five aborted fetuses from the same litter, and 17 sera collected from sows infected or normal were submitted to the Pathology Division of the National Veterinary Research and Quarantine Service for diagnostic investigation. Grossly, hepatomegaly and splenomegaly were observed in sows. Multiple necrotic foci were scattered in the lungs, liver, spleen, and lymph nodes. Microscopically, multifocal necrotizing lesions and protozoan tachyzoites were present in the lesions. Tachyzoites of Toxoplasma (T.) gondii were detected immunohistochemically. Latex agglutination showed that the sera of 7 of 17 (41.2%) sows were positive for antibody to T. gondii. The disease outbreak in this herd was diagnosed as epizootic toxoplasmosis. To our knowledge, this is the first report of porcine toxoplasmosis with a high abortion rate and sow mortality in Korea.     

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。     

Mic1-3 Knockout Toxoplasma gondii is a good candidate for a vaccine against T. gondii-induced abortion in sheep

This study assessed the effectiveness of a mutant strain of Toxoplasma gondii (RH strain) lacking the mic1 and mic3 genes (Mic1-3KO) against Toxoplasma abortion in sheep. Ewes were inoculated subcutaneously with 10⁵ Mic1-3KO tachyzoïtes in three independent experiments. Following vaccination, Mic1-3KO induced a mild febrile response and serum IgG antibodies, which persisted throughout the experiments. Tissue cysts formed in the sheep, but were not, under our experimental conditions, infectious when given orally. Ewes were mated two months after vaccination and were orally challenged with the PRU strain of T. gondii at mid-gestation (400 oocysts in Experiments 1 and 2; 100 oocysts in Experiment 3). Challenge of vaccinated pregnant ewes resulted in a slight febrile response, whereas unvaccinated ewes developed a more severe, characteristic febrile response of longer duration. After challenge, all unvaccinated ewes aborted whereas 62%, 91% and 64% (Experiments 1, 2 and 3 respectively) of the lambs from vaccinated ewes were viable, with no clinical signs of infection. Mic1-3KO was as effective as S48, the strain used as a live vaccine for sheep (Toxovax^®). A dose of 10⁵ Mic1-3KO tachyzoites was sufficient to induce protection (versus a dose of 2 × 10⁶). Both subcutaneous and intraperitoneal injections were effective. Moreover, preliminary results showed the potential of Mic1-3KO to reduce the development of tissue cysts in lambs born to vaccinated ewes. This study demonstrates that Mic1-3KO is a potent vaccine candidate.     

Biologic and molecular characterization of Toxoplasma gondii isolates from pigs from Portugal

Little is known of Toxoplasma gondii infections in animals in Portugal. In the present paper, we report the first isolation of viable T. gondii from pigs in Portugal. Antibodies to T. gondii were found in 52 (15.6%) of 333 pigs prior to slaughter using the modified agglutination test (MAT) at a serum dilution of 1:20. Attempts were made to isolate T. gondii from 37 seropositive pigs. Samples of brain and/or heart from each pig were digested in acid pepsin, and bioassayed into mice. Viable T. gondii was isolated from 15 pigs. Restriction fragment length polymorphism on products of SAG2 locus amplified by PCR and microsatellite analysis revealed that 11 isolates were Type II and four were Type III. The results indicate that phenotypically and genetically T. gondii are similar to isolates from pigs from the U.S.     

Population genetics of Toxoplasma gondii: new perspectives from parasite genotypes in wildlife

Toxoplasma gondii, a zoonotic protozoal parasite, is well-known for its global distribution and its ability to infect virtually all warm-blooded vertebrates. Nonetheless, attempts to describe the population structure of T. gondii have been primarily limited to samples isolated from humans and domesticated animals. More recent studies, however, have made efforts to characterize T. gondii isolates from a wider range of host species and geographic locales. These findings have dramatically changed our perception of the extent of genetic diversity in T. gondii and the relative roles of sexual recombination and clonal propagation in the parasite's lifecycle. In particular, identification of novel, disease-causing T. gondii strains in wildlife has raised concerns from both a conservation and public health perspective as to whether distinct domestic and sylvatic parasite gene pools exist. If so, overlap of these cycles may represent regions of high probability of disease emergence. Here, we attempt to answer these key questions by reviewing recent studies of T. gondii infections in wildlife, highlighting those which have advanced our understanding of the genetic diversity and population biology of this important zoonotic pathogen.     

弓形虫MIC10基因敲除株的构建及鉴定

为了解弓形虫MIC10基因特性,利用CRISPR/Cas9技术构建了弓形虫MIC10基因敲除虫株,通过乙胺嘧啶药物筛选、GFP绿色荧光观察及96孔板筛选阳性敲除虫株,并进行体外和小鼠体内PCR鉴定MIC10基因进一步确定单克隆敲除虫株。结果表明,加入乙胺嘧啶后MIC10基因敲除株能够正常生长,在荧光显微镜下观察敲除株呈现绿色荧光,在96孔板的单孔中筛选出唯一绿色荧光虫体;经体外和小鼠体内PCR鉴定MIC10基因,敲除虫株扩增出1 000 bp的外源片段,未扩增出MIC10基因片段,而在对照组RH虫株扩增出263 bp MIC10基因片段,未扩增出1 000 bp外源片段;说明本试验成功构建并筛选出MIC10基因敲除的单克隆虫株,暂命名为弓形虫KO-MIC10-RH。本试验为弓形虫MIC10基因敲除株的致病性研究奠定基础。     

The protective effect of a Toxoplasma gondii SAG1 plasmid DNA vaccine in mice is enhanced with IL-18

More effective vaccines against Toxoplasma gondii may contribute to the control of this pathogen that has major veterinary and public health significance. In this study, two recombinant plasmids pcDNA/TgSAG1 and pVAX/mIL-18 containing T. gondii SAG1 (TgSAG1) and murine cytokine interleukin-18 (IL-18) were evaluated for their ability to protect mice against T. gondii challenge. Mice were given two intramuscular immunizations 3 weeks apart, and challenged with T. gondii 3 weeks later. All animals vaccinated with pcDNA/TgSAG1 alone or with pVAX/mIL-18 developed specific anti-TLA (T. gondii lysate antigen) antibodies and specific lymphocyte proliferative responses. Co-injection of pVAX/mIL-18 significantly increased the production of IFN-γ and IL-2. Further, challenge experiments showed that co-immunization with pVAX/mIL-18 significantly (P < 0.05) increased the survival rate (60%), compared with pcDNA/TgSAG1 alone (40%). Therefore, codelivery of the IL-18-secreting plasmid potentiates the induction and maintenance of the type 1 helper T-cell immune response and may be a potent strategy for enhancing the protective efficacy of vaccines against T. gondii.     

Prevalence of IgG antibodies to Encephalitozoon cuniculi and Toxoplasma gondii in cats with and without chronic kidney disease from Virginia

Kidney disease is a common and serious condition in domestic cats. There are numerous causes of kidney disease including parasitic infection. Encephalitozoon cuniculi is a microsporidian parasite that develops in the kidneys of rabbits and causes chronic renal disease. Little has been reported concerning E. cuniculi in cats and no serological studies on this parasite in cats have been conducted in the United States to date. The present study explored the possibility that E. cuniculi is an unrecognized contributor to the high prevalence of kidney disease observed in cats. A serological survey was conducted to determine the prevalence of IgG antibodies to spores of E. cuniculi in cats with and without a diagnosis of chronic kidney disease (CKD) according to the International Renal Interest Society (IRIS) staging system. Likewise, samples were examined for IgG antibodies to Toxoplasma gondii, a common well studied protozoan of cats. Plasma and sera were obtained from 232 feline patients at the Virginia-Maryland Regional College of Veterinary Medicine teaching hospital. With the investigators blinded to the renal status of test subjects, samples were screened via indirect immunofluorescent antibody assay (IFA). Thirty-six of the 232 cats met the IRIS staging system criteria for CKD. Antibodies to E. cuniculi were found in 15 of the 232 samples, which included 4 of the 36 cats with CKD. Sera from cats serologically positive to E. cuniculi did not react to spores of E. intestinalis or E. hellem when examined in the IFA. Antibodies to T. gondii were found in 63 of the 232 samples, which included 10 of the 36 cats with CKD. The prevalence of antibodies in cats with CKD to either protozoan was not significantly different (P>0.05) from the cats without CKD in the study. Collectively the results do not support the hypothesis that either E. cuniculi or T. gondii play a significant etiologic role in the occurrence or progression of CKD in cats.     

Evaluation of the diagnostic accuracy of the modified agglutination test (MAT) and an indirect ELISA for the detection of serum antibodies against Toxoplasma gondii in sheep through Bayesian approaches

The diagnostic accuracies of the modified agglutination test (MAT) and indirect ELISA test for the detection of serum antibodies against Toxoplasma gondii in sheep were evaluated through Bayesian approaches on two populations of sheep created from three different groups of animals (T. gondii-aborted ewes, colostrums-deprived newborn lambs, and ewe-lambs and adult ewes with unknown T. gondii infection status). Tests showed a high degree of agreement (kappa statistic = 0.93; 95% confidence interval = 0.87, 0.98) and a significant specificity (Sp) correlation (gamma(Sp) = 0.26; 95% credibility interval = 0.017, 0.61). When prior information was used for all unknown parameters the posterior medians for the sensitivity (Se) and Sp of the MAT and ELISA were, respectively, 92.6% (95% credibility interval = 85.2, 96.9), 95.5% (89.9, 98.7), 90.5% (83.4, 95.6), and 97.8% (94.2, 99.5). These estimates remained similar when uninformative priors were included. The Se estimates of the MAT and ELISA were higher than those obtained on pigs in other study using the same approach (Se = 80.6% and Sp = 89.5% for the MAT, and Se = 71.5% and Sp = 85.5% for the ELISA [Georgiadis, M.P., Wesley, O.J., Gardner, I.A., Singh, R., 2003. Correlation-adjusted estimation of sensitivity and specificity of two diagnostic tests. Appl. Stat. 52, 63-78]. This finding supported the believe that test performances may vary when applied on different animal species. Thus, if these tests are planned to be used on animal species other than sheep or pigs, their diagnostic accuracy should be re-assessed to prevent biased inferences from their results.     

Cross-sectional survey on Toxoplasma gondii infection in cattle, sheep and pigs in Serbia: seroprevalence and risk factors

Toxoplasmosis is a globally distributed zoonosis with a clinical impact in the unborn fetus and in the immunosuppressed individual. In Serbia, studies of risk factors for Toxoplasma gondii infection in humans have shown that the relatively high prevalence is associated mainly with consumption of undercooked meat and/or meat products. However, data on T. gondii infection in domestic animals mostly used for human consumption are scarce. We thus conducted a cross-sectional survey on the seroprevalence of T. gondii infection in a representative sample of cattle, sheep and pigs from different regions of Serbia between June 2002 and June 2003, and analyzed the main risk factors associated with the infection. Sera from 611 cattle (yearlings and adults of both sexes), 511 ewes, and 605 pigs (market-weight and sows), were examined for T. gondii antibodies by the modified agglutination test. The seroprevalences determined were 76.3% in cattle, 84.5% in sheep and 28.9% in pigs. The antibody levels ranged from 1:25 to 1:400 in cattle, and up to 1:25,600 in sheep and to 1:12,800 in pigs. Among the seropositive, the proportion of high antibody levels (> or =1:1600), suggestive of acute infection, was 10% in sheep, and 4% in pigs. Possible association of the infection with biologically plausible risk factors including gender, age, herd size/farm type, type of housing, feeding practices and region, was analyzed by univariate analysis, and variables significant at P< or =0.1 were included in multivariate logistic regression models. The results showed that risk factors for cattle were small herd size (odds ratio, OR=2.19, 95% confidence interval, CI=1.28-3.75, P=0.004) and farm location in Western Serbia (OR=2.04, 95% CI=1.10-3.79, P=0.024), while housing in stables with access to outside pens was protective (OR=0.37, 95% CI=0.21-0.67, P=0.001). In sheep, an increased risk of infection was found in ewes from state-owned flocks (OR=4.18, 95% CI=2.18-8.00, P<0.001) vs. private flocks, and, interestingly, also in those from Western Serbia (OR=4.66, 95% CI=1.18-18.32, P=0.028). In pigs, the risk of infection was highly increased in adult animals (OR=3.87, 95% CI=2.6-5.76, P<0.001), as well as in those from finishing type farms (OR=3.96, 95% CI=1.97-7.94, P<0.001). In addition to providing data on the current T. gondii seroprevalence in meat animals in Serbia, the results of this study show the main risk factors associated with infection, thereby pointing to the type of preventive measures to reduce T. gondii infection.     

Majority of T. gondii seropositive chickens (Gallus domesticus) in Central Ethiopia carries the infective parasite

Background

The prevalence of Toxoplasma gondii in free range chickens is a good indicator of the prevalence of T. gondii oocysts in the environment. The aim of this study was to isolate T. gondii parasites from heart and brain of seropositive free range (FR) chickens.

Findings

Isolation of T. gondii from pooled heart and brain of 41 direct agglutination test (DAT) positive (≥1:40) free range chickens (Gallus domesticus) was carried out by bioassay in mice. T. gondii specific antibodies in mice were assayed by DAT and microscopy was employed for detection and enumeration of brain tissue cysts. Overall, bioassay was positive in 29 (70.7%) chicken samples. T. gondii tissue cysts were isolated from 59% (24/41) of bioassayed chickens: from 2 of 7 chickens with a titer of 1: ≤ 60, 2 of 5 with titer 1: 180, 6 of 8 with titer 1: 540, 10 of 15 with titer 1: 1620, 1 of 2 with titer 1: 6000, 2 of 3 with titer 1:18000, 1 of 1 with titer 1:54000. None of the isolates was pathogenic for mice. Tissue cysts were detected from 61% of seropositive mice (DAT ≥ 1:40). Generally, tissue cyst counts per brain of mouse were low (mean: 132.7 ± 84.4; range: 47–352).

Conclusions

Majority of T. gondii seropositive chickens (Gallus domesticus) in Central. Ethiopia carries the infective parasite. Tissues from the free range chicken might be a source infection for animals and humans.     

桦褐孔菌多糖对弓形虫感染孕鼠生殖器官及其胎鼠发育的影响

为探讨桦褐孔菌多糖对弓形虫感染孕鼠生殖器官及其胎鼠发育的影响,采用弓形虫RH株感染雌性性成熟小鼠,以蒿甲醚为阳性对照组,桦褐孔菌多糖为试验组,并设立阴性组和模型组.观察孕鼠胎儿的死胎散、体重、体长等发育指标,同时记录剖检前各组孕鼠死亡数和受孕数,并对孕鼠的生殖器官进行病理组织观察.经统计分析,桦褐孔菌多糖组与模型组在孕鼠死亡率和受孕率方面差异极显著(P＜0.01);在死胎率、活胎鼠平均体重、活胎鼠平均体长方面差异也极显著(P＜0.01);而两药物组仅在受孕率方面显著差异(P＜0.05),其孕鼠生殖器官的病理组织仅呈轻微的炎症变化.表明桦褐孔菌多糖对弓形虫感染孕鼠的胎鼠发育具有保护作用;并可明显缓解弓形虫感染对孕鼠生殖机能的损害.     

Seroepidemiology of Neospora caninum and Toxoplasma gondii in cattle and water buffaloes (Bubalus bubalis) in the People's Republic of China

A seroepidemiological survey of Neospora caninum and Toxoplasma gondii in cattle and water buffaloes was carried out in the People's Republic of China. Serum samples were obtained from dairy (n=262, 9 herds in 9 provinces) and beef cattle (n=10, 1 herd) and water buffaloes (n=40) in China. All sera were tested for antibodies to N. caninum and T. gondii by an enzyme-linked immunosorbent assay (ELISA) and an indirect agglutination test (IAT), respectively. The overall seroprevalence of N. caninum in dairy cattle was 17.2% (45/262), and the herds seroprevalence of N. caninum was 88.9% (8/9), and antibodies to T. gondii were present in 6 cows (2.3%). None of the cows had antibodies against both T. gondii and N. caninum. Antibodies to T. gondii or N. caninum were not found in beef cattle or water buffaloes. The seroprevalence of N. caninum in aborting cows (20.2%) was higher than that in non-aborting cows (16.6%) with an odds ratio of 1.26 (95% CI, 0.54-2.95), but the difference was not statistically significant (P>0.05). There was no apparent association of N. caninum seropositivity with age or number of pregnancies. This is the first report on the seroprevalence of N. caninum in cattle and water buffaloes in China.     

Genotyping studies of Toxoplasma gondii isolates from Africa revealed that the archetypal clonal lineages predominate as in North America and Europe

Until recently, Toxoplasma gondii was considered to be clonal with very little genetic variability. Recent studies indicate that T. gondii isolates from Brazil are genetically and biologically different from T. gondii isolates from USA and Europe. However, little is known of the genetics of T. gondii strains from Africa. In this study, we genotyped 19 T. gondii isolates from chickens from six African countries (Egypt, Kenya, Nigeria, Congo, Mali, and Burkina Fasco) using 10 PCR-RFLP markers (SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico). The results revealed four genotypes. Thirteen isolates belong to the Type III lineage, five isolates have Type II alleles at all loci except apico and they belong to the Type II lineage. One isolate from Nigeria had atypical genotype. In general, these isolates were mostly clonal Type III and II strains that predominate in North American and European. DNA sequencing at several loci for representative isolates confirmed the results of PCR-RFLP genotyping. Taken together with recent studies of T. gondii isolates from Africa, it is clear that the three clonal lineages (Types I, II and III) predominate not only in North America and Europe, but also in Africa.