Identification of potential apicoplast associated therapeutic targets in human and animal pathogen Toxoplasma gondii ME49

Toxoplasma gondii ME49 is an obligatory intracellular apicomplexa parasite that causes toxoplasmosis in humans, domesticated and wild animals. Waterborne outbreaks of acute toxoplasmosis worldwide reinforce the transmission of Toxoplasma gondii ME49 to humans through contaminated water and may have a greater epidemiological impact than previously believed. In the quest for drug and vaccine target identification subtractive genomics involving subtraction between the host and pathogen genome has been implemented for enlisting essential pathogen specific proteins. Using this approach, our analysis on both human and Toxoplasma gondii ME49 reveals that out of 7987 protein coding sequences of the pathogen, 950 represent essential non human-homologous proteins. Subcellular localization prediction & comparative-biochemical pathway analysis of these essential proteins gives a list of apicoplast-associated proteins having unique pathogen-specific metabolic pathway. These apicoplast-associated enzymes involved in fatty acid biosynthesis pathway of Toxoplasma gondii ME49, may be used as potential drug targets, as the pathway is vital for the protozoan's survival. Structure prediction of drug target proteins was done using fold based recognition method. Screening of the functional inhibitors against these novel targets may result in discovery of novel therapeutic compounds that can be effective against Toxoplasma gondii ME49. ABBREVIATIONS: DEG - Database of Essential Gene, KEGG - Kyoto Encyclopaedia of Genes and Genomes, KAAS - KEGG Automated Annotation Server, PFP - Protein Function Prediction, COG - Cluster of Orthologous Genes.     

中药大蒜素体外抗弓形虫效应及其机制的研究

目的 观察大蒜提取物体外抗弓形虫的作用效果及其机制.方法 将弓形虫RH株速殖子与兔肾细胞共同培养,加入不同浓度的大蒜素(实验组)和磺胺嘧啶(阳性对照组),培养不同时间后取出细胞,固定染色后观察细胞感染率及每个纳虫泡中弓形虫速殖子的数量;采用MTT比色法观察大蒜素对弓形虫速殖子侵袭细胞及其正常细胞增殖的影响;采用台盼兰着色法观察大蒜素对弓形虫速殖子活性的影响;采用TUNEL末端标记法检测弓形虫速殖子凋亡率,对药物的效应和机制进行探讨.结果 (1)10～80 μg/ml的大蒜素能抗弓形虫的感染,呈现剂量依赖性,与时间无明显的相关性.(2)40 μg/ml、80 μg/ml的大蒜素不能抑制细胞的增殖,对细胞无明显的毒副作用;160 μg/ml的大蒜素对细胞有明显的毒副作用.(3)大蒜素80 μg/ml时,台盼兰着色率最高,弓形虫的活力最低.(4)大蒜素80 μg/ml的浓度时,弓形虫速殖子凋亡率最高.结论 大蒜素可以抑制弓形虫速殖子的活力、对宿主细胞的感染力、在细胞内的增殖,无明显的毒副作用,最适宜浓度为80 μg/ml.大蒜素是一种良好的抗弓形虫药物,诱导弓形虫速殖子凋亡是其抗弓形虫机制之一.     

刚地弓形虫基因工程疫苗

弓形虫疫苗是预防弓形虫感染的最有效手段之一。随着生物学研究技术的不断进步,弓形虫疫苗的研究也不断深入完善。本文介绍了近年来发展起来的基因工程疫苗的最新研究现状及进展,以期望在对现有研究成果总结的基础上,为大家以后的研究提供新的视角和方向。     

Toxoplasma gondii infection in humans and animals in the United States

This paper reviews clinical and asymptomatic Toxoplasma gondii infection in humans and other animals in the USA. Seroprevalence of T. gondii in humans and pigs is declining. Modes of transmission, epidemiology and environmental contamination with oocysts on land and sea are discussed.     

Seroprevalence of Toxoplasma gondii in wild toque macaques (Macaca sinica) at Polonnaruwa, Sri Lanka

From a natural population that inhabits the dry evergreen forest at Polonnaruwa, serum samples of 170 toque macaques were examined for antibodies to Toxoplasma gondii by the modified agglutination test. Of these, 21 (12%) were found with titers of 1:16 in 9, 1:32 in 9, 1:256 in 1, 1:1,024 in 1, and 1:4,096 in 1. There was no evidence of maternal transmission of antibodies or congenital toxoplasmosis. None of the infected macaques died within 1 yr after sampling. Toxoplasma gondii infection was closely linked to human environments where domestic cats were common. Macaques having frequent contact with human settlements showed a significantly greater (P < 0.0001) prevalence (19% infected) than macaques restricted to forest habitat, none of which was infected. Although infection with T. gondii has been noted in several species of Asian primates, this is the first report of T. gondii antibodies in toque macaques (Macaca sinica) that are endemic to the island of Sri Lanka.     

Speculation on possible life cycles for the clonal lineages in the genus toxoplasma

Recent evidence suggests that the strains currently classified in the genus Toxoplasma, ie. within the species Toxoplasma gondii, may actually comprise at least two clonal lineages correlated with their virulence in mice. Here, Alan Johnson reviews these data in the context of evolution and speciation within the genus, and raises hypotheses on how the virulent lineage may undergo an asexual life cycle in nature, similar to that found for the very closely related coccidian, Neospora camnum. The putative vertical transmission life cycle of this mouse virulent lineage of T. gondii could involve passage to the foetus late in pregnancy, or transmission in milk to the neonate after birth.     

Molecular and biologic characteristics of Toxoplasma gondii isolates from wildlife in the United States

Toxoplasma gondii isolates can be grouped into 3 genetic lineages. Type I isolates are considered more virulent in outbred mice and have been isolated predominantly from clinical cases of human toxoplasmosis, whereas types II and III isolates are considered less virulent for mice and are found in humans and food animals. Little is known of genotypes of T. gondii isolates from wild animals. In the present report, genotypes of isolates of T. gondii from wildlife in the United States are described. Sera from wildlife were tested for antibodies to T. gondii with the modified agglutination test, and tissues from animals with titers of 1:25 (seropositive) were bioassayed in mice. Toxoplasma gondii was isolated from the hearts of 21 of 34 seropositive white-tailed deer (Odocoileus virginianus) from Mississippi and from 7 of 29 raccoons (Procyon lotor); 5 of 6 bobcats (Lynx rufus); and the gray fox (Urocyon cinereoargenteus), red fox (Vulpes vulpes), and coyote (Canis latrans) from Georgia. Toxoplasma gondii was also isolated from 7 of 10 seropositive black bears (Ursus americanus) from Pennsylvania by bioassay in cats. All 3 genotypes of T. gondii based on the SAG2 locus were circulating among wildlife.     

Signaling during the invasion of host cells by Toxoplasma gondii

Invasion of host cells is essential for the pathogenicity of Toxoplasma gondii. This review examines the signal transduction pathways that lead to the internalization of T. gondii. We demonstrate that extra- and intracellular Ca(2+) mobilization, Ca(2+)-calmodulin complex and phospholipase A(2) activities are required for T. gondii entry. T. gondii also causes the activation of mitogen-activated protein kinase in infected cells and modifies its ionic environment during its intracellular state. Thus, many of the signaling systems found in other eukaryotes are operative in Toxoplasma invasion.     

Toxoplasma gondii infections in red-tailed hawks inoculated orally with tissue cysts

The response to inoculation of Toxoplasma gondii tissue cysts was examined in 3 red-tailed hawks (Buteo jamaicensis). One hawk (hawk 1) was inoculated orally with 3.000 tissue cysts of the GT-1 isolate of T. gondii and 2 hawks (hawks 2 and 3) each were inoculated orally with 12,000 tissue cysts of a mixture of 8 isolates of T. gondii. None of the hawks developed clinical signs of toxoplasmosis. Serum antibodies were measured with the modified direct agglutination test using formalin-fixed tachyzoites. Hawk 1 had a titer of 1:40 prior to inoculation and did not have an increase in titer during the study. Hawks 2 and 3 had titers of 1:5 and 1:10, respectively, prior to inoculation, and both had increased titers (titers greater than or equal to 1:60) by 1 wk postinoculation and remained T. gondii antibody positive throughout the 10 wk of the study. Toxoplasma gondii was isolated from the heart and breast muscle of hawk 1. The biologic behavior of this T. gondii isolate was different from the 1 inoculated, and it probably represents a prior natural infection. Toxoplasma gondii was isolated from the brain, heart, breast muscle, and a mixture of gizzard and proventriculus from hawk 2 and from breast muscle of hawk 3. Toxoplasma gondii was not isolated from the eye, lung, liver, kidney, or spleen of any red-tailed hawk.     

Does vertical transmission contribute to the prevalence of toxoplasmosis?

Toxoplasma gondii is a ubiquitous parasite with a widespread distribution both in terms of geographical and host range. Although the definitive host is the cat, it is also a major health hazard to domestic animals and humans. Three routes of transmission are recognised (infection from the cat, carnivory and congenital transmission). We aimed to assess the relative importance of congenital transmission, using sheep as a model system, due to the lack of carnivory. We report, using PCR as a diagnostic tool, that congenital transmission occurs with high frequency (69%). If transmission from oocysts was important in sheep, we would expect sheep reared under the same environmental conditions (i.e. a single farm) to have a random distribution of Toxoplasma infection. Using breeding records in conjunction with PCR, some families were found to have high Toxoplasma prevalence and abortion while others were free of Toxoplasma infection and abortion (P < 0.01). This supports the notion that Toxoplasma may be transmitted vertically. In humans, we conducted a similar study and showed that Toxoplasma was transmitted from mother to baby in 19.8% of cases. Vertical transmission in Toxoplasma may be more important than previously thought and this knowledge should be considered in any eradication strategies.     

Earthworms as paratenic hosts of toxoplasmosis in eastern barred bandicoots in Tasmania

An experimental feeding study was designed to assess the role of earthworms in the transmission of Toxoplasma gondii infection to eastern barred bandicoots (Perameles gunnii). Six animals with no agglutinating antibodies to T. gondii were fed artificially cultured earthworms that had been maintained in autoclaved nutrient-enriched soil. Two animals were given earthworms that had been maintained in soil contaminated with T. gondii oocysts (P89/VEG strain); two animals were fed on earthworms, which initially had been exposed to soil containing T. gondii oocysts then transferred through three changes of sterile soil; two control bandicoots were fed earthworms maintained in sterile soil. Both bandicoots fed earthworms maintained in T. gondii contaminated soil died 11 and 14 days after feeding. The necropsy findings were consistent with acute toxoplasmosis. Bandicoots fed earthworms exposed to oocysts but then transferred through changes of sterilized soil remained healthy as did control animals. All surviving animals remained seronegative over the 6 wk observation period after feeding. These findings confirm that earthworms, a major component of the natural diet of P. gunnii, can transmit T. gondii infection. It appears that oocysts present in the alimentary tracts of the worms, rather than infective stages of T. gondii in worm somatic tissues, are responsible for these infections.     

Isolation and genetic characterization of Toxoplasma gondii from striped dolphin (Stenella coeruleoalba) from Costa Rica

Toxoplasma gondii infection in marine mammals is of interest because of mortality and mode of transmission. It has been suggested that marine mammals become infected with T. gondii oocysts washed from land to the sea. We report the isolation and genetic characterization of viable T. gondii from a striped dolphin (Stenella coeruleoalba), the first time from this host. An adult female dolphin was found stranded on the Pacific Coast of Costa Rica, and the animal died the next day. The dolphin had a high (1:6400) antibody titer to T. gondii in the modified agglutination test. Severe nonsuppurative meningoencephalomyelitis was found in its brain and spinal cord, but T. gondii was not found in histological sections of the dolphin. Portions of its brain and the heart were bioassayed in mice for the isolation of T. gondii. Viable T. gondii was isolated from the brain, but not from the heart, of the dolphin. A cat fed mice infected with the dolphin isolate (designated TgSdCol) shed oocysts. Genomic DNA from tachyzoites of this isolate was used for genotyping at 10 genetic loci, including SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico, and this TgSdCo1 isolate was found to be Type II.     

Development and evaluation of an enzyme-linked immunosorbent assay with recombinant SAG2 for diagnosis of Toxoplasma gondii infection in cats

Cats are pivotal in the transmission of Toxoplasma gondii. To develop a sensitive and specific serodiagnostic method for feline toxoplasmosis, surface antigen 2 (SAG2) of T. gondii was expressed in Escherichia coli and its diagnostic potential evaluated in an enzyme-linked immunosorbent assay (ELISA). The ELISA with recombinant SAG2 (rSAG2) was able to differentiate very clearly between sera from cats experimentally infected with T. gondii and sera from normal cats. Serum samples collected from domestic cats in Japan were investigated by the ELISA, and the results were compared with those of a commercially available latex agglutination test (LAT) kit. Of the 192 samples screened, 42 (21.9%) were positive by ELISA. Among the 42 ELISA-positive samples, 39 were positive by LAT. There was a significant correlation between ELISA and LAT titers. All the 150 ELISA-negative samples were negative by LAT. These results indicate that the ELISA with rSAG2 expressed in E. coli should be a useful method for detection of T. gondii infection in cats.     

Isolation and genotyping of Toxoplasma gondii from free-ranging chickens from Argentina

The prevalence of Toxoplasma gondii in free-ranging chickens can be considered a good indicator of the prevalence of T. gondii oocysts in the environment because chickens feed from the ground. In the present study, prevalence of T. gondii in 29 free-range chickens (Gallus domesticus) from Argentina was investigated. Blood, heart, and brain from each chicken were examined for T. gondii infection. Antibodies to T. gondii, assayed with the modified agglutination test (MAT), were found in 19 of 29 (65.5%) chickens. Hearts and brains of seropositive (MAT > or = 1:5) chickens were bioassayed in mice. Toxoplasma gondii was isolated from 9 of 19 seropositive chickens. Genotyping of chicken isolates of T. gondii using the SAG2 locus indicated that 1 was type I, 1 was type II, and 7 were type III. This is the first report of isolation of T. gondii from chickens from Argentina.     

弓形虫GRA6蛋白和P30蛋白的融合表达、纯化及抗原性分析

利用基因工程技术制备抗原性好的弓形虫GRA6蛋白和P30蛋白的融合蛋白,并用作抗原检测弓形虫抗体。根据弓形虫GRA6蛋白和P30蛋白的氨基酸序列,通过计算机分析,筛选出其中较强的抗原决定簇。用PCR方法分别扩增含抗原决定簇的基因片段。将这两个基因片段克隆至同一质粒pET28a(+)内,表达一个融合蛋白。将重组质粒转化大肠杆菌BL21(DE3),筛选表达该融合蛋白的工程菌。纯化表达的融合蛋白,用已知的6份抗弓形虫IgM阳性血清和大量正常人血清,ELISA法检测纯化融合蛋白的抗原性和特异性。获得了高效表达含弓形虫GRA6蛋白和P30蛋白抗原表位的工程菌,表达的融合蛋白约占菌体蛋白总量的25%。纯化获得了表达的融合蛋白,该蛋白有较好的抗原性和特异性。表达的弓形虫GRA6和P30融合蛋白可用做抗原检测弓形虫抗体,用于临床及孕妇检测,对优生优育有较大意义。     

宠物弓形虫的感染及其防控

弓形虫病是一种世界性分布的人兽共患寄生虫病,对人类,尤其是妇女、儿童危害很大,估计全世界有1/3人受到该病的威胁。孕妇感染弓形虫后导致早产、流产、胎儿发育畸形;弓形虫是免疫功能低下患者的主要死亡原因之一。犬、猫是弓形虫的中间宿主和终末宿主,是人类感染弓形虫的主要来源。随着我国经济的迅速发展和人民生活水平的不断提高,城市中饲养犬、猫作为宠物的人越来越多,人、宠物间的亲密接触增加了弓形虫病传播给人的机会。加强对宠物犬、猫弓形虫病的研究及防控势在必行。本文就弓形虫的危害、宠物犬猫弓形虫感染及其防控措施作以综述。     

Characterisation of Toxoplasma gondii isolates using polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) of the non-coding Toxoplasma gondii (TGR)-gene sequences

The Toxoplasma gondii (TGR) genes constitute a family of non-coding sequences, three of which have been previously described as possible tools for typing of Toxoplasma gondii isolates. We obtained new isolates of T. gondii from domestic and wild animals, and used these to evaluate the possibility of using TGR gene variants as markers to distinguish among T. gondii isolates from different animals and different geographical sources.Based on the band patterns obtained by restriction fragment length polymorphism (RFLP) analysis of the polymerase chain reaction (PCR) amplified TGR sequences, the T. gondii isolates could be separated into seven groups. Sequencing the amplified products showed that at least 20 TGR sequences not hitherto described had been found, demonstrating that the TGR gene family comprises a large number of different yet highly homologous sequences. Each isolate had its own unique TGR sequence. The TGR gene family therefore seems a promising target for typing individual T. gondii isolates and for studying the genetic distance between two isolates, which can be used for tracing routes of infection.     

Biologic and molecular characteristics of Toxoplasma gondii isolates from striped skunk (Mephitis mephitis), Canada goose (Branta canadensis), black-winged lory (Eos cyanogenia), and cats (Felis catus)

Toxoplasma gondii isolates can be grouped into 3 genetic lineages. Type I isolates are considered virulent to outbred mice, whereas Type II and III isolates are not. In the present report, viable T. gondii was isolated for the first time from striped skunk (Mephitis mephitis), Canada goose (Branta canadensis), and black-winged lory (Eos cyanogenia). For the isolation of T. gondii, tissues were bioassayed in mice, and genotyping was based on the SAG2 locus. Toxoplasma gondii was isolated from 3 of 6 skunks, 1 of 4 Canada geese, and 2 of 2 feral cats (Felis catus) from Mississippi. All donor animals were asymptomatic. Viable T. gondii was also isolated from 5 of 5 lories that had died of acute toxoplasmosis in an aviary in South Carolina. Genotypes of T. gondii isolates were Type III (all skunks, lories, and the goose) and Type II (both cats). All 5 Type III isolates from birds and 2 of the 3 isolates from skunks were mouse virulent.     

Toxoplasma gondii: from animals to humans

Toxoplasmosis is one of the more common parasitic zoonoses world-wide. Its causative agent, Toxoplasma gondii, is a facultatively heteroxenous, polyxenous protozoon that has developed several potential routes of transmission within and between different host species. If first contracted during pregnancy, T. gondii may be transmitted vertically by tachyzoites that are passed to the foetus via the placenta. Horizontal transmission of T. gondii may involve three life-cycle stages, i.e. ingesting infectious oocysts from the environment or ingesting tissue cysts or tachyzoites which are contained in meat or primary offal (viscera) of many different animals. Transmission may also occur via tachyzoites contained in blood products, tissue transplants, or unpasteurised milk. However, it is not known which of these routes is more important epidemiologically. In the past, the consumption of raw or undercooked meat, in particular of pigs and sheep, has been regarded as a major route of transmission to humans. However, recent studies showed that the prevalence of T. gondii in meat-producing animals decreased considerably over the past 20 years in areas with intensive farm management. For example, in several countries of the European Union prevalences of T. gondii in fattening pigs are now <1%. Considering these data it is unlikely that pork is still a major source of infection for humans in these countries. However, it is likely that the major routes of transmission are different in human populations with differences in culture and eating habits. In the Americas, recent outbreaks of acute toxoplasmosis in humans have been associated with oocyst contamination of the environment. Therefore, future epidemiological studies on T. gondii infections should consider the role of oocysts as potential sources of infection for humans, and methods to monitor these are currently being developed. This review presents recent epidemiological data on T. gondii, hypotheses on the major routes of transmission to humans in different populations, and preventive measures that may reduce the risk of contracting a primary infection during pregnancy.     

Survival of Toxoplasma gondii oocysts in Eastern oysters (Crassostrea virginica)

Toxoplasma gondii has recently been recognized to be widely prevalent in the marine environment. It has previously been determined that Eastern oysters (Crassostrea virginica) can remove sporulated T. gondii oocysts from seawater and that oocysts retain their infectivity for mice. This study examined the long-term survival of T. gondii oocysts in oysters and examined how efficient oysters were at removing oocysts from seawater. Oysters in 76-L aquaria (15 oysters per aquarium) were exposed to 1 x 10(6) oocysts for 24 hr and examined at intervals up to 85 days postexposure (PE). Ninety percent (9 of 10) of these oysters were positive on day 1 PE using mouse bioassay. Tissue cysts were observed in 1 of 2 mice fed tissue from oysters exposed 21 days previously. Toxoplasma gondii antibodies were found in 2 of 3 mice fed oysters that had been exposed 85 days previously. In another study, groups of 10 oysters in 76-L aquaria were exposed to 1 x 10(5), 5 x 10(4), or 1 x 10(4) sporulated T. gondii oocysts for 24 hr and then processed for bioassay in mice. All oysters exposed to 1 x 10(5) oocysts were infected, and 60% of oysters exposed to 5 x 10(4) oocysts were positive when fed to mice. The studies with exposure to 1 x 10(4) oocysts were repeated twice, and 10 and 25% of oysters were positive when fed to mice. These studies indicate that T. gondii can survive for several months in oysters and that oysters can readily remove T. gondii oocysts from seawater. Infected filter feeders may serve as a source of T. gondii for marine mammals and possibly humans.