Secretion of multi-protein migratory complex induced by Toxoplasma gondii infection in macrophages involves the uPA/uPAR activation system

Toxoplasmosis is a world wide spread zoonosis caused by Toxoplasma gondii, an obligate intracellular parasite that is able to disseminate into deep tissues and cross biological barriers, reaching immunoprivileged sites such as the brain and retina. The parasite is able to infect macrophages and dendritic cells for dispersal throughout the body. However, the molecular mechanisms or outcomes of the subversion of the host cell are largely unknown. Recently our group established that metalloproteinases are involved in migration of infected macrophages. Herein, we evaluated the recruitment of host invasive machinery components in T. gondii infected murine macrophages. We showed by immunoprecipitation assays that MMP-9, CD44 TIMP-1 and uPAR were secreted as a multi-protein complex by infected macrophages. Zymographic analysis revealed that MMP-9 was present in its pro- and active form. Moreover, inhibition of uPA/uPAR pathway by PAI-1 decreased secretion of MMP-9 active forms, as well those associated to uPAR and TIMP-1, but not to CD44. Data presented here suggest that MMP-9 is secreted as a multiprotein complex by T. gondii infected macrophages, similar to that observed in metastatic cells. We further speculate that uPA/uPAR system is involved in the expression/secretion of complexes containing active MMP-9 forms.     

应用巢式PCR-杂交技术检出人血样及动物排泄物中弓形虫DNA

弓形虫是一种专性细胞内寄生的原虫，引起人兽共患疾病-弓形虫病。本文报道了一种基于PCR技术检测宠物弓形虫的方法，该方法的特点是通过检测宠物排泄物中是否存在弓形虫包囊，从而诊断宠物是否为真实的传染源。由于宠物的排泄物容易采集，避免了复杂的采样程序，方便于检测。     

弓形虫病疫苗研究进展

综述了弓形虫病减毒和灭活疫苗、基因工程亚单位疫苗、核酸疫苗及活载体疫苗的研究进展。弓形虫是专性细胞内寄生原虫,全球分布,能引起人兽共患的弓形虫病。弓形虫病疫苗的研制对于弓形虫病的防治工作有着极其重要的意义。     

Toxoplasma gondii induction of interleukin-12 is associated with acute virulence in mice and depends on the host genotype

The intracellular parasite Toxoplasma gondii can influence host resistance by modulating immune functions in various cell types. The stimulation of interleukin (IL)-12 production in macrophages, dendritic cells and neutrophils by T. gondii has been implicated to be important for skewing anti-parasite immunity early after infection as well as in mediating the pathologic effects induced by the parasite. The present study demonstrates secretion of IL-12 p40 and the bioactive p70 heterodimer by inflammatory macrophages following exposure to live Toxoplasma or tachyzoite lysate. Parasite induction of IL-12 occurred in a dose-dependent manner. Predigestion of T. gondii lysate with proteinase K abrogated its IL-12 inducing activity, thus indicating that a parasite protein(s) triggers this response. Macrophages from various mouse inbred strains showed a differential responsiveness: cells from T. gondii-susceptible mice released more IL-12 upon toxoplasmic challenge than those from resistant mice, although the infection rate and intracellular parasite growth were similar. In triggering macrophage production of IL-12, tachyzoites proved superior to bradyzoites prepared from the same T. gondii isolate. Furthermore, parasites of a mouse-virulent isolate became less efficient inducers of IL-12 following attenuation. The parallel loss in macrophage stimulation in vitro and acute virulence in vivo suggests a linkage of both parasite capacities. Together with the correlation on host side between the genotype-dependent mouse susceptibility to infection and cellular responsiveness to the parasite trigger, these findings indicate that an overproduction of parasite-induced IL-12 might represent a basic mechanism of T. gondii pathogenicity.     

Enrofloxacin is able to control Toxoplasma gondii infection in both in vitro and in vivo experimental models

Currently, toxoplasmosis is treated with sulfadiazine and pyrimethamine. However, this treatment presents several adverse side effects; thus, there is a critical need for the development and evaluation of new drugs, which do not present the same problems of the standard therapy. Enrofloxacin is a fluoroquinolone antibiotic known to control infection against several bacteria in veterinary medicine. Recently, this drug has demonstrated protective effects against protozoan parasites such as Neospora caninum. The present study aimed to determine the effect of enrofloxacin in the control of Toxoplasma gondii infection. For this purpose, human foreskin fibroblast (HFF) cells were infected with T. gondii RH strain and treated with sulfadiazine, penicillin/streptomycin, pyrimethamine, or enrofloxacin. Following treatment, we analyzed the infection index, parasite intracellular proliferation and the number of plaques. Additionally, tissue parasitism and histological changes were investigated in the brain of Calomys callosus that were infected with T. gondii (ME49 strain) and treated with either sulfadiazine or enrofloxacin. Enrofloxacin was able to reduce the infection index, intracellular proliferation and the number of plaques in HFF cells infected by T. gondii in comparison with untreated or penicillin/streptomycin-treated ones. Enrofloxacin was more protective against T. gondii in HFF infected cells than sulfadiazine treatment (P<0.001). In addition, pyrimethamine, enrofloxacin or the associations of sulfadiazine plus pyrimethamine, enrofloxacin plus sulfadiazine or enrofloxacin plus pyrimethamine-treatments were able to reduce the plaque numbers in HFF cells infected by T. gondii when compared to medium, penicillin/streptomycin or sulfadiazine alone. In vivo experiments demonstrated that enrofloxacin diminished significantly the tissue parasitism as well as the inflammatory alterations in the brain of C. callosus infected with T. gondii when compared with untreated animals. Based on our findings, it can be concluded that enrofloxacin is a potential alternative drug for the treatment of toxoplasmosis.     

Host cholesterol synthesis contributes to growth of intracellular Toxoplasma gondii in macrophages

The intracellular protozoan Toxoplasma gondii lacks the ability to synthesize sterol and scavenges cholesterol from the low-density lipoprotein receptor (LDLR) pathway of its host to facilitate replication. Sterol biosynthesis inhibitors, however, have a demonstrated anti-Toxoplasma effect. In this study, we examined the host mevalonate pathway as a novel source of cholesterol for T. gondii and its effects on parasite growth in macrophages. Parasite growth did not significantly change in the absence of LDLR or when LDL was exogenously supplemented. Lovastatin and compactin, both inhibitors of hydroxymethylglutaryl-CoA (HMG-CoA) reductase in the mevalonate pathway, significantly inhibited T. gondii growth in both wild-type and LDLR-knockout macrophages. Parasite growth was also suppressed by squalestatin, an inhibitor of squalene synthase, despite mevalonate producing isoprenoid intermediates in host cells. The present study demonstrates that lovastatin, compactin and squalestatin have anti-Toxoplasma activities and that the host cholesterol synthesis may contribute to parasite growth in macrophages.     

抗弓形虫药物研究进展

刚地弓形虫(Toxoplasma gondii)是一种呈世界范围分布的人兽共患寄生原虫,给人类生活和畜牧业生产带来极大的危害,然而至今仍没有理想的治疗弓形虫病的药物。近年来,研究人员主要从干扰弓形虫的叶酸代谢、检测抗其他病原微生物和寄生虫药物的抗弓形虫活性,以及针对弓形虫独特的生理结构和生化反应路径进行药物设计,开展抗弓形虫药物的研究,取得了许多重要的成果。随着人们对弓形虫研究的不断深入,对其分子生物学特性会有更加全面的了解,将给新的药物靶标的发现和特效药物的研发提供重要的理论依据。     

Toxoplasma gondii rhomboid protein 1 (TgROM1) is a potential vaccine candidate against toxoplasmosis

Infection with the intracellular protozoan parasite Toxoplasma gondii causes serious public health problems and is of great economic importance worldwide. The rhomboid proteins which are responsible for adhesion and invasion of host cells have been suggested as vaccine candidates against toxoplasmosis. A DNA vaccine (pVAX-ROM1) encoding T. gondii rhomboid protein 1 (TgROM1) gene was constructed and the immune response and protective efficacy of this vaccine against lethal challenge in BALB/c mice were evaluated. The results indicated that specific antibody and lymphocyte proliferative responses were elicited in mice receiving pVAX-ROM1. The production levels of IFN-γ, IL-2, IL-4, and IL-10, as well as the percentage of CD4(+) cells in mice vaccinated with pVAX-ROM1 were significantly increased respectively, compared to controls receiving either pVAX1 alone or PBS. After lethal challenge, the mice immunized with pVAX-ROM1 showed an increased survival time compared with the mice in the controls. Our data suggested that a DNA vaccine pVAX-ROM1 encoding T. gondii rhomboid protein 1 triggered strong humoral and cellular responses, and prolonged survival time against T. gondii infection in BALB/c mice.     

Longitudinal study on the seroprevalence of Toxoplasma gondii infection in four German pig breeding and raising farms

There are very few current data on the prevalence of Toxoplasma (T.) gondii in German pig farms. Consequently a reliable risk assessment of human Toxoplasmosis caused by ingesting raw or improperly cooked pork and pork products is not available. The aim of this study was to show current data on T. gondii prevalence in German pig farms. In four pig farms with different management systems (three conventional, one organic) 100 animals each were selected and tested for T. gondii antibodies. The test was done four times during the period from birth to slaughtering. In one farm 20 mother sows were tested additionally. The slaughtered pigs from conventional farms showed seroprevalences between 0 and 15.2% (mean value 5.6%). At the organic system T. gondii antibodies were not detected. All slaughtered seropositive pigs (6 months old) were tested negatively at the age of 9 weeks, but shortly after birth high titres of T. gondii antibodies had been detected in the same animals. Comparing the results gained in different seasons significantly more pigs were found to be infected during the autumn/winter than in the spring/summer period. In order to assess the current risk of Toxoplasmosis more pig farms should be tested. From the point of view of consumer protection the detection of highly infected pig herds is necessary.     

Biology and epidemiology of Toxoplasma gondii in man and animals

Toxoplasma gondii is a coccidian parasite which utilizes felids as definitive hosts, and which has an unusually wide intermediate host range. The parasite was initially described by Nicolle and Manceaux in 1908 from the rodent, Ctenodactylus gundi. Infection with T. gondii is one of the most common parasitic infections of man and other warm-blooded animals. It has been found worldwide from Alaska to Australia. Nearly one-third of humanity has been exposed to this parasite; serologic surveys indicate that T. gondii infections are common in wild carnivores, including pigs, bears, felids, fox, raccoons, and skunks. Clinical and subclinical toxoplasmosis has been reported from wild cervids, ungulates, marsupials, monkeys, and marine mammals. Southern sea otter populations have been severely impacted by Toxoplasma infections.     

Study of Toxoplasma infection in Brazilian wild mammals: serological evidence in Dasypus novemcinctus Linnaeus, 1758 and Euphractus sexcinctus Wagler, 1830

Toxoplasmosis is a worldwide distributed zoonosis that affects man and most warm-blooded animals, with a great economic impact in animal and public health. Serum samples from nine 9-banded armadillos, three 6-banded armadillos, three coatimundis, two opossums and one nutria were submitted for anti-Toxoplasma gondii antibody detection by means of a modified direct agglutination method. Encephalic tissue of three 6-banded armadillos, one 9-banded armadillo, one coatimundi and one nutria were digested in acid pepsin solution and inoculated into Swiss mice for parasite isolation. Only one serum sample from a nine-banded armadillo and two from six-banded armadillos reacted producing titers equal to 256, 512 and 512, respectively. T. gondii was isolated in two 6-banded armadillos, one of which was not positive in the serological test.     

A decade of discoveries in veterinary protozoology changes our concept of "subclinical" toxoplasmosis

One of the most compelling topics to emerge from the last decade of veterinary protozoology is disease caused by a zoonotic pathogen, Toxoplasma gondii, in otherwise healthy people. These findings may catch the health professions by surprise, because veterinary and medical courses and textbooks typically emphasize that T. gondii infections are subclinical, unless acquired in utero or the patient has a serious immunosuppressive condition. Nevertheless, numerous reports in the last decade associate toxoplasmosis with lymphadenopathy, fever, weakness and debilitation, ophthalmitis, and severe multisystemic infections in people who do not have immunosuppressive conditions. Toxoplasmosis in rodents causes altered behavior, and similar mental aberrations are coming to light in humans; recent studies associate T. gondii infection with personality shifts and increased likelihood of reduced intelligence or schizophrenia. These conditions reduce the quality of life of individuals, and may exact a significant economic burden upon society. Of course, toxoplasmosis continues to cause serious conditions in AIDS patients and congenitally infected people, as well as abortions and encephalitis in domestic and wild animals. Environmental contamination is heavy enough to extend into marine wildlife. It is time for the health professions to amend teaching curricula regarding T. gondii. Veterinary parasitologists should lead the way in developing methods to reduce the prevalence of T. gondii in food animals. Public health policies should prohibit the practice of allowing pet cats to roam. Organizations and individuals that feed feral cats are unwittingly contributing to the dissemination of T. gondii, by sustaining artificially dense populations of a definitive host of this protozoal parasite.     

High tissue burden of Toxoplasma gondii is the hallmark of acute virulence in mice

Toxoplasma gondii virulence is commonly determined by mortality rate of infected mice. Limited data showed that virulent T. gondii strains had increased parasite growth in mice compared to that of less virulent strains. To determine if this is a common phenomenon for a variety of strains and to develop an alternative assay to test acute virulence in mice, we measured parasite burdens in experimentally infected outbred CD-1 mice for 19 T. gondii isolates, in which the virulence phenotypes had previously been determined by mortality assay. Our results showed that parasite concentrations in spleen tissues were two orders of magnitude higher in the virulent than the intermediately and non-virulent isolates at day 7 post infection. In competition assays, mice inoculated with mixed tachyzoites of virulent and intermediately virulent strains or virulent and non-virulent strains showed that the former always reached a higher concentration at day 7 post infection. In mixed infection of intermediate and non-virulent strains, both strains were detectable in mice at day 7 post infection. In conclusion, our data showed that the virulence of T. gondii can be predicted by parasite load in the spleen tissue of infected mice at 7 days post infection, providing an alternative method to determine virulence of Toxoplasma.     

Toxoplasmosis

Toxoplasma gondii is a protozoan parasite that infects most species of warm-blooded animals, including human beings. Infection in cats is of particular interest because Felidae (domestic and wild) are the only known definitive hosts for T. gondii and thus are the main reservoirs of infection. Clinical and public health aspects of T. gondii infections in cats and dogs are discussed in this article.     

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.     

孕妇弓形虫病防治研究进展

弓形虫病是一种世界性分布的人兽共患寄生虫病,在人和动物中广泛传播。孕妇感染弓形虫后多表现为隐性感染,无明显的临床表现,但该虫可通过垂直传播影响胎儿发育,造成早产、流产、畸形和死胎等不良妊娠现象。论文对弓形虫病流行病学、感染因素、免疫学、诊断和治疗等方面阐述弓形虫感染对孕妇的影响,为孕妇弓形虫病的防治提供参考。     

Evaluation of Toxoplasma gondii and Neospora caninum infections in sheep from Uberlândia, Minas Gerais State, Brazil, by different serological methods

Toxoplasmosis and neosporosis have been recognized as economically important diseases with considerable impact on the livestock industry. Considering the scarce information on the occurrence of Toxoplasma gondii and Neospora caninum infections in sheep from Uberlandia, Minas Gerais State, Brazil, this study aimed to investigate the frequency of antibodies against these parasites in sheep sera from this region by using different serological methods. A total of 155 sheep serum samples were analyzed by the indirect fluorescence antibody test (IFAT) and enzyme-linked immunosorbent assay (ELISA) for the detection of IgG against T. gondii and N. caninum. Seroreactivity by IFAT showed 80% of samples with titers between 512 and 2048 for T. gondii (cutoff ≥ 64) and 78% presenting titers between 50 and 200 for N. caninum (cutoff ≥ 50). Seroreactivity by ELISA showed 75% of samples with ELISA index (EI) between 2.0 and 3.0 for T. gondii (cutoff ≥ 1.3) and 54% presenting EI between 1.3 and 2.0 for N. caninum (cut off ≥ 1.3). Discordant results by both tests were analyzed by immunoblot, resulting in a total seropositivity of 61% for T. gondii and 23% for N. caninum, with 41% to T. gondii only, 3% to N. caninum only, and 20% to both parasites. There was a significant positive association between seropositivity to T. gondii and age over one year (P<0.001), but such association was not found for N. caninum infection. In conclusion, as T. gondii and N. caninum infections are simultaneously present in sheep flocks of this region, it should be emphasized the importance to carry out a regular monitoring of Toxoplasma infection due to its high prevalence, its zoonotic potential and induction of reproductive disorders leading to economic losses. For neosporosis, sheep farmers should be instructed about the presence of the parasite in the flock, its risk factors and potential abortifacient role in sheep. Differential flock management could be valuable tool to establish the association of serological positivity and reproductive disease induced by N. caninum in sheep.     

Evaluation of three novel azasterols against Toxoplasma gondii

Previous studies from our group have demonstrated the high susceptibility of Toxoplasma gondii tachyzoites to the sterol analogues 22,26-azasterol and 24,25-(R,S)-epiminolanosterol. In this work we present data on testing in vitro three novel azasterols as potential agents for the treatment of toxoplasmosis. The three compounds inhibited parasite growth at micromolar concentrations, in a dose-dependent manner. Electron microscopy analysis of intracellular tachyzoites after treatment with the most effective compound showed drastic mitochondrion swelling associated with the appearance of an electron-lucent matrix and disrupted cristae. Parasite lysis also took place. The appearance of electron dense cytoplasmic structures similar to amylopectin granules distributed throughout the parasite suggests that azasterols might be inducing differentiation of those tachyzoites which were not lysed to the bradyzoite stage.     

Toxoplasma gondii and ovine toxoplasmosis: new aspects of an old story

Ovine toxoplasmosis, caused by the intracellular protozoan parasite Toxoplasma gondii, was first described in 1954 and while the incidence of ovine infection is difficult to define, it has been reported that in the UK it is responsible for between 1 and 2% of neonatal losses per annum. Recent reports have suggested that sheep persistently infected with T. gondii may pass infection to the fetus in subsequent pregnancies more readily than previously thought. These data show a high proportion of both successful and failed pregnancies in sheep to be positive by PCR for T. gondii with a tendency for samples from certain genetic lines of Charollais sheep more likely to be positive than samples from other lines, suggesting that some sheep have a particular genetic susceptibility to T. gondii.     

Seroprevalence of Toxoplasma gondii infection in domestic goats in Satun Province, Thailand

Goats are important domestic animals in the south of Thailand due to the minimal cost of rearing and maintaining them, and their production of both meat and milk. Toxoplasmosis is one of the most threatening parasitic zoonoses and the causative agent Toxoplasma gondii uses a wide range of warm-blooded intermediate hosts including the goat. The objective of this study was to assess the seroprevalence of antibodies to T. gondii in goats of Satun Province in Thailand. A total of 631 goat sera were examined for antibodies against toxoplasmosis with commercial latex agglutination test kits (Toxocheck-MT 'Eiken'). Of these, 176 (27.9%) were found to be positive to T. gondii; antibody titers ranged from 1:64 to 1:4096 (1:64 cut-off). Female goats were 1.73 times more likely than male to be seropositive (odds ratio [OR]=1.73; 95% confidential interval [CI]=1.11, 2.73). Dairy goats were more likely to be seropositive than meat goats (OR=1.36; 95% CI=0.84, 2.20). Goats were infected with T. gondii with acquisition of age because older goats were more likely to be seropositive than young goats under 1-year-old (for 1-2 years, OR=19.6; 95% CI=0.92, 4.15, for >2 years, OR=2.70; 95% CI=1.26, 5.80). The high seroprevalence of T. gondii antibodies found in the present study suggested widespread exposure of goats in Satun Province to T. gondii.