Cyst Burden Research Articles

GRA7 provides protective immunity in cocktail DNA vaccines against Toxoplasma gondii

In a previous study, single-gene vaccination with GRA1, GRA7 or ROP2 was shown to elicit partial protection against Toxoplasma gondii. In this study, the contribution of each antigen in the evoked humoral and cellular immune responses was evaluated after vaccination with plasmid mixtures containing GRA1, GRA7 and ROP2. Cocktail DNA vaccinated mice developed high antibody titers against the antigens from two-gene DNA vaccine cocktails, but lower titres when immunized with the three-gene cocktail. High numbers of IFN-gamma secreting splenocytes were generated predominantly against GRA7. Brain cyst burden was reduced by 81% in mice vaccinated with the three-gene mixture and they were completely protected against acute toxoplasmosis. Similar high levels of brain cyst reductions were obtained after vaccination with cocktails composed of GRA1 and GRA7 (89% reduction), or GRA7 and ROP2 (79% reduction), but not with the cocktail composed of GRA1 and ROP2. In low dose single-gene vaccinations, IFN-gamma and strong protection could only be elicited by GRA7. Hence, the presence of GRA7 in the DNA vaccine formulation was important for optimal protection and this was correlated with GRA7-specific IFN-gamma production. We propose GRA7 as a main component in cocktail DNA vaccines for vaccination against T. gondii.

Azithromycin macrolids with a potent activity on chronic experimental toxopiasmosis

Objective: To assess the efficacy of Azithromycin, a macrolids antibiotic, againsst experimentally induced toxopiasmosis in marine model Design: Experimental study. Setting: Department of Microbiology, Nineveh College of Medicine, from October 2004 to November 2005. Results: The efficacy of Azithromycin against Toxoplasma gondii was examined in murine models of infection with a moderately virulent strain. Forty white Albino mice inoculated by mouth with 2x 10 cysts, 20 mice were treated with Azithromycin at dose of 250 mg/kg /day for 90 days. Drug treatment was initiated 30 days after infection (chronic infection). Treatment with Azithromycin significantly enhanced survival and reduced the brain cyst burden as compared with untreated (control) group of mice. Since 80% of treated mice were cornp>letely cured from toxopiasmosis as their brain were free of Toxoplasma tissue cysts either microscopically or by bioassay. Conclusion: The results warrant clinical evaluation of Azithromycin in the treatment of toxopiasmosis in both immunocompetent and immunocompromised patients.

Polycystic Liver Disease: Multimodality Imaging for Complications and Transplant Evaluation

Polycystic liver disease (PLD) is usually associated with polycystic kidney disease but may also occur as an isolated finding in a rarer genetically distinct disease. In either case, the cyst burden will progress over time and, in rare cases, may affect liver function or become symptomatic due to massive hepatomegaly. The character, distribution, location, and size of hepatic cysts are important. Computed tomography, magnetic resonance imaging, or ultrasonography may provide the surgeon with valuable preoperative information, such as the location of infected or hemorrhagic cysts that may be responsible for symptoms. Less invasive cyst aspiration or fenestration may provide temporary relief from dominant or symptomatic cysts, but these cysts will recur in up to 75% of patients. Cyst fenestration with partial hepatic resection and liver transplantation are two therapies that provide more permanent resolution of symptoms in patients with extensive hepatic involvement. However, the higher risk of complications associated with more aggressive surgical therapy must be considered when determining the appropriate therapy for a given patient. Knowledge of the cyst patterns and available treatment options in patients with PLD will help the radiologist provide the referring clinician with important information for therapeutic decision making.

Neurocysticercosis

Since international travel has become widespread, clinicians increasingly have been confronted with the diagnosis of symptomatic and incidental neurocysticercosis. The difficulty with managing this disease lies in its varied clinical presentations. The treatment of neurocysticercosis depends upon anatomic locations, stages of cysts, and overall cyst burden. Despite recent clinical and laboratory advances, experts are divided on many key management questions. This article reviews the pathophysiology and experimental data underlying treatment recommendations for this emerging and complex condition.

SECONDARY TRANSMISSION IN PORCINE CYSTICERCOSIS: DESCRIPTION AND THEIR POTENTIAL IMPLICATIONS FOR CONTROL SUSTAINABILITY

Taenia solium taeniasis/cysticercosis is one of few potentially eradicable infectious diseases and is the target of control programs in several countries. The larval stage of this zoonotic cestode invades the human brain and is responsible for most cases of adult-onset epilepsy in the world. The pig is the natural intermediate host, harboring the larvae or cysticerci. Our current understanding of the life cycle implicates humans as the only definitive host and tapeworm carrier (developing taeniasis) and thus the sole source of infective eggs that are responsible for cysticercosis in both human and pigs through oral-fecal transmission. Here we show evidence of an alternative pig-to-pig route of transmission, previously not suspected to exist. In a series of four experiments, naive sentinel pigs were exposed to pigs that had been infected orally with tapeworm segments (containing infective eggs) and moved to a clean environment. Consistently in all four experiments, at least one of the sentinel pigs became seropositive or infected with parasite cysts with much lower cyst burdens than did primarily infected animals. Second-hand transmission of Taenia solium eggs could explain the overdispersed pattern of porcine cysticercosis, with few pigs harboring heavy parasite burdens and many more harboring small numbers of parasites. This route of transmission opens new avenues for consideration with respect to control strategies.

Effectiveness of spiramycin in murine models of acute and chronic toxoplasmosis

The antitoxoplasmic activity of spiramycin (SPI) was evaluated in murine models of infection using a type-1 (RH) or type-2 (Me49) strain of Toxoplasma gondii. In mice infected with 10 2 tachyzoites of the RH strain, treatment with 100 and 200 mg SPI/kg/day had only a limited effect; despite some dose-dependent prolongation of survival, it was unable to protect mice against death. In contrast, in acute infection induced by peroral inoculation of 10, but not 20, cysts of the Me49 strain, a 3-week course of 100 mg SPI/kg/day and a 4-week course of 200 mg/kg/day significantly enhanced protection and markedly reduced brain cyst burdens at 6 months post infection (p.i.). In chronic infection established by inoculation of 10 cysts 3 months previously, a 3-week course of 200 mg SPI/kg/day resulted in significantly decreased brain cyst burdens compared with controls, both 2 weeks after treatment cessation and by 6 months p.i. Although a favourable effect on chronic infection may be specific for mice, these data merit investigation, since they may have clinical ramifications.

Production of the Main Surface Antigen of <I>Toxoplasma gondii</I> in Tobacco Leaves and Analysis of Its Antigenicity and Immunogenicity

We adapted a previously described Agrobacterium-mediated transient expression system to test the expression level of three constructs carrying the surface antigen 1 (SAG1) of Toxoplasma gondii. Two constructs were based in a Potato virus X (PVX) amplicon. In one of them, the PVX movement protein genes were replaced by the sag1 gene. In the other, the sag1 gene was placed under the control of an additional coat protein subgenomic promoter. In the third construct, the sag1 gene was fused to an apoplastic peptide signal under the CaMV 35S promoter. Western blot analysis of leaf extracts infiltrated with each construct revealed a protein of 35 kDa. SAG1 accumulation in leaves ranged from 0.1 to 0.06% of total soluble protein (equivalent to 10 microg and 6 microg of SAG1 per gram of fresh leaf tissue, respectively). Three of five human seropositive samples reacted with tobacco-expressed SAG1 in Western blot analysis. The C3H mice were immunized with SAG-expressing leaf extracts and perorally challenged with a nonlethal dose of the T. gondii Me49 strain. Mice vaccinated with SAG1 showed significantly lower brain cyst burdens compared to those from the control group. Immunization with SAG1-expressing leaves elicited a specific humoral response with predominant participation of type IgG2a. In conclusion, a functional SAG1 version could be transiently expressed in tobacco leaves.

The Expression of Lactate Dehydrogenase Is Important for the Cell Cycle of Toxoplasma gondii

In Toxoplasma gondii, lactate dehydrogenase is encoded by two independent and developmentally regulated genes LDH1 and LDH2. These genes and their products have been implicated in the control of a metabolic flux during parasite differentiation. To investigate the significance of LDH1 and LDH2 in this process, we generated stable transgenic parasite lines in which the expression of these two expressed isoforms of lactate dehydrogenase was knocked down in a stage-specific manner. These LDH knockdown parasites exhibited variable growth rates in either the tachyzoite or the bradyzoite stage, as compared with the parental parasites. Their differentiation processes were impaired when the parasites were grown under in vitro conditions. In vivo studies in a murine model system revealed that tachyzoites of these parasite lines were unable to form significant numbers of tissue cysts and to establish a chronic infection. Most importantly, all mice that were initially infected with tachyzoites of either of the four LDH knockdown lines survived a subsequent challenge with tachyzoites of the parental parasites (10(4)), a dose that usually causes 100% mortality, suggesting that live vaccination of mice with the LDH knockdown tachyzoites can confer protection against T. gondii. Thus, we conclude that LDH expression is essential for parasite differentiation. The knockdown of LDH1 and LDH2 expression gave rise to virulence-attenuated parasites that were unable to exhibit a significant brain cyst burden in a murine model of chronic infection.

Effects of snail size on encystment of Echinostoma caproni in juvenile Biomphalaria glabrata (NMRI strain) and observations on the survival of infected snails.

The effects of snail size on encystment of Echinostoma caproni cercariae in neonatal and juvenile Biomphalaria glabrata (NMRI strain) snails were studied. Encystment in neonatal (0.7-1.1 mm shell diameter) and juvenile (2-3 mm shell diameter) snails was compared 24 h post-infection (PI) following individual exposure of snails of each size to 1, 5, 10, 25 and 50 cercariae. Significantly more cysts were recovered from juveniles exposed to 1, 5, 10 and 50 cercariae than from neonatals with comparable exposure. Size of B. glabrata was a major factor in determining cyst burden in this planorbid. Survival of infected versus uninfected neonatals and juveniles was also examined for 7 days. Neonatals exposed to 10 cercariae showed a significant decrease in survival at 3, 6 and 7 days PI when compared to the uninfected controls. There was no significant decrease in the survival of juveniles exposed to 10 cercariae compared to uninfected controls at any time point. Snail size was a factor in mortality associated with echinostome cercarial penetration and encystment.

Effects of snail size and diet on encystment of Echinostoma caproni cercariae in juvenile Helisoma trivolvis (Colorado strain) and observations on survival of infected snails.

The effects of snail size and diet on encystment of Echinostoma caproni cercariae in juvenile Helisoma trivolvis (Colorado strain) snails were studied. Encystment in neonatal (<1-mm shell diameter) and juvenile (2- to 3-mm shell diameter) snails was compared 24 hr postinfection (PI) after individual exposure of snails of each size to 1, 5, 10, 25, or 50 cercariae. Significantly more cysts were recovered from juvenile snails exposed to 10, 25, or 50 cercariae than from neonatals with comparable exposure. The maximum number of cysts recovered from juveniles exposed to 50 cercariae was 42, compared with a maximum of 15 cysts in neonatals comparably exposed. Size of H. trivolvis was a major factor in determining cyst burden in this planorbid. A diet of either Romaine lettuce leaf or hen's egg yolk did not have a significant effect on the number of cysts recovered at 24 hr PI from juvenile snails exposed to 25 or 75 cercariae. Survival of infected versus uninfected neonatals was also examined for 7 days. Neonatals exposed to 10 cercariae showed a significant decrease in survival at 6 and 7 days PI when compared with uninfected controls.

DNA vaccination with the immunodominant tachyzoite surface antigen (SAG-1) protects against adult acquired Toxoplasma gondii infection but does not prevent maternofoetal transmission

We examined the ability of a DNA vaccine comprising the Toxoplasma gondii tachyzoite immunodominant surface antigen, SAG-1, to both protect adult BALB/c mice against infection with the avirulent Beverly type-2 strain of T. gondii and also to inhibit the incidence of congenital disease. Vaccination induced an enhanced type-1 immune response as measured by IgG2a antibody production and increased splenocyte IFN-γ production. Vaccination also limited disease following infection via either the oral or peritoneal routes as measured by mortality, pathology or brain cyst burden. While vaccination with plasmid alone also increased splenocyte IFN-γ production, this afforded no protection and following infection mortality rates and cyst burden counts were similar in this group to that of non-vaccinated animals. Although, vaccination with SAG-1 DNA did protect against adult acquired T. gondii infection, it did not prevent maternofoetal transmission in previously vaccinated dams infected during pregnancy. The results indicate differences in the protective mechanisms operating between adult acquired disease and congenital transmission and have significant implications for future vaccine development.

A GRA1 DNA vaccine primes cytolytic CD8(+) T cells to control acute Toxoplasma gondii infection.

Protective immunity against Toxoplasma gondii is known to be mediated mainly by T lymphocytes and gamma interferon (IFN-gamma). The contribution of CD4(+) and CD8(+) T-lymphocyte subsets to protective immune responses against T. gondii infection, triggered by a GRA1 (p24) DNA vaccine, was assessed in this study. In vitro T-cell depletion experiments indicated that both CD4(+) and CD8(+) T-cell subsets produced IFN-gamma upon restimulation with a T. gondii lysate. In addition, the GRA1 DNA vaccine elicited CD8(+) T cells that were shown to have cytolytic activity against parasite-infected target cells and a GRA1-transfected cell line. C3H mice immunized with the GRA1 DNA vaccine showed 75 to 100% protection, while 0 to 25% of the mice immunized with the empty control vector survived challenge with T. gondii cysts. In vivo T-cell depletion experiments indicated that CD8(+) T cells were essential for the survival of GRA1-vaccinated C3H mice during the acute phase of T. gondii infection, while depletion of CD4(+) T cells led to an increase in brain cyst burden during the chronic phase of infection.

Efficacy of atovaquone combined with clindamycin against murine infection with a cystogenic (Me49) strain of Toxoplasma gondii

The efficacy of atovaquone (ATO) combined with clindamycin (CLI) against Toxoplasma gondii was examined in murine models of infection with a mouse-non-virulent (Me49) strain. Swiss-Webster mice inoculated by mouth with 10 or 20 cysts were treated with ATO and CLI alone or combined at dosages of ATO 5-100 and CLI 25-400 mg/kg/day for 2-4 weeks. Drug treatment was initiated (i) day 4 post-infection (acute infection), (ii) 3 months post-infection (chronic infection) and (iii) following a 2-3 week course of treatment with dexamethasone (DXM) alone or combined with cortisone-acetate (CA) introduced 3 months post-infection (reactivated toxoplasmosis). In acute infection, whereas treatment with any drug or drug combination significantly enhanced survival and reduced the brain cyst burden, in mice treated with ATO alone or combined with CLI, the cyst counts were significantly lower than in mice treated with CLI alone. In chronic infection, the decrease in the cyst burden observed 2 weeks after treatment with either drug alone was significant only in mice treated with the combined drugs. Most importantly, in reactivated toxoplasmosis, whereas an effect for the combined drugs was shown in mice suppressed with both DXM alone and combined with CA, in mice pre-treated with DXM a 3 week course of ATO > or = 25 and CLI 50 mg/kg/day significantly increased survival and markedly decreased the cyst burden. The latter effect was long-term, since the cyst burdens in treated mice continued to decrease up to 3 months later, whereas they increased in the untreated mice. The results warrant clinical evaluation of the combination of ATO and CLI in the treatment of toxoplasmosis in both immunocompetent and, more importantly, immunosuppressed patients.

Cyst burden in the brains of Wistar rats fed Toxoplasma oocysts.

Six strains of Toxoplasma oocysts were used to infect groups of 4-24 Wistar rats, with each rat being fed 10(1)-10(4) oocysts from a single strain. After 2 months, the rats were killed, their brains screened for Toxoplasma cysts and then bioassayed in mice if negative. Toxoplasma was either observed in the form of brain cysts or was recovered using the bioassay, from 113 out of 138 (82%) rat brains. As few as ten oocysts were capable of initiating a brain infection that lasted for at least 2 months in eight of the nine rats inoculated. However, judging from bioassay 10(2)-10(4) oocysts did not give rise to progressively higher rates of infection. Brain cysts were seen in only 68 of 138 rats (49%). The number of Toxoplasma cysts formed in the brains of rats was generally in the order of tens to hundreds. The frequency of infection in the brains with Toxoplasma and the number of brain cysts formed appeared to be influenced by the individual resistance of the rats as well as by the doses of oocysts and the Toxoplasma strains used. The information gathered is considered to be a basis for a rat model of immunity against acquired toxoplasmosis.

Deaths associated with cysticercosis

There is significant concern about an increased risk of death in patients with neurocysticercosis who receive antihelminthic agents. To clarify the risk factors for death associated with cysticercosis, the authors identified all cases of mortality over a 5-year period in patients in whom cysticercosis was diagnosed and who were discharged from a major metropolitan medical center. In addition, they reviewed the literature to delineate risk factors associated with cysticercosis-related death, concluding that the risk of death is highest in patients with heavy cyst burdens and those surgically treated for intracranial hypertension. Death is rare in properly selected patients with parenchymal disease in whom the cyst burden is low and intracranial hypertension is absent. The most common causes of death appear to be 1) intracranial hypertension in surgically treated patients and 2) shunt infection.

Genetic analysis of tachyzoite to bradyzoite differentiation mutants in Toxoplasma gondii reveals a hierarchy of gene induction.

Developmental switching in Toxoplasma gondii, from the virulent tachyzoite to the relatively quiescent bradyzoite stage, is responsible for disease propagation and reactivation. We have generated tachyzoite to bradyzoite differentiation (Tbd-) mutants in T. gondii and used these in combination with a cDNA microarray to identify developmental pathways in bradyzoite formation. Four independently generated Tbd- mutants were analysed and had defects in bradyzoite development in response to multiple bradyzoite-inducing conditions, a stable phenotype after in vivo passages and a markedly reduced brain cyst burden in a murine model of chronic infection. Transcriptional profiles of mutant and wild-type parasites, growing under bradyzoite conditions, revealed a hierarchy of developmentally regulated genes, including many bradyzoite-induced genes whose transcripts were reduced in all mutants. A set of non-developmentally regulated genes whose transcripts were less abundant in Tbd- mutants were also identified. These may represent genes that mediate downstream effects and/or whose expression is dependent on the same transcription factors as the bradyzoite-induced set. Using these data, we have generated a model of transcription regulation during bradyzoite development in T. gondii. Our approach shows the utility of this system as a model to study developmental biology in single-celled eukaryotes including protozoa and fungi.

Cutting edge: IL-12 is required for the maintenance of IFN-gamma production in T cells mediating chronic resistance to the intracellular pathogen, Toxoplasma gondii.

IL-12 is required for the development of IFN-gamma-dependent resistance to intracellular pathogens but is not thought to play a major role in its maintenance. To directly assess the requirement for continuous IL-12 signaling in long-term cell-mediated immunity, recombinant cytokine was transiently administered to IL-12 p40-deficient mice during the first 2 wk of infection with the intracellular pathogen Toxoplasma gondii. As expected, these animals survived the acute phase and established chronic infections. However, 4-6 wk after IL-12 withdrawal, the mice exhibited increased brain cyst burdens and succumbed to toxoplasmic encephalitis. Reactivation was associated with a loss of T-dependent IFN-gamma production without a concomitant increase in Th2 cytokine expression. Importantly, parasite Ag-induced IFN-gamma synthesis by purified T cells from these animals could be restored by in vitro exposure to IL-12. These results argue that endogenous IL-12 is required for the long-term maintenance of IFN-gamma-dependent resistance against intracellular pathogens.

Host-parasite relationships between Echinostoma caproni and RAG-2-deficient mice.

The RAG-2-deficient mouse, a strain of genetically altered mice lacking B- and T-lymphocytes, was used as a host for Echinostoma caproni. In all, 12 male RAG mice were exposed to 25 cysts each, and 12 served as uninfected controls. Mice were necropsied at 2 and 3 weeks postinfection (p.i.). The mean number+/-SE (9.7+/-2.4) of worms recovered from infected mice at 2 weeks p.i. was not significantly different from that recovered at 3 weeks p.i. (6.5+/-2.2). The intestinal circumference of infected RAG mice was significantly greater than that of the controls at 2 and 3 weeks p.i. A significant goblet cell hyperplasia occurred at 2 weeks p.i., but the response was not effective in eliminating worms from the RAG mice. The effect of a high cyst burden was examined by exposure of 8 RAG and 8 ICR mice to 100 cysts each. The body length and area and the oral sucker area of worms grown in RAG mice were significantly greater than those of worms grown in ICR mice. Worm recovery at up to 3 months p.i. was examined in RAG mice exposed to 25 cysts and necropsied every 2 weeks p.i. The mean worm recovery recorded at 2 weeks p.i. was significantly greater than that noted at 12 weeks p.i., at which time worm rejection from the RAG mouse host first occurred. The RAG mouse is a useful host for studies on E. caproni in a murine host that lacks B- and T-lymphocytes.

Limitations of current diagnostic procedures for the diagnosis of Taenia solium cysticercosis in rural pigs

The aim of the present study was to evaluate diagnostic procedures for porcine cysticercosis. Sera were obtained from 32 pigs reared in commercial farms, 47 pigs before and after experimental infection, 42 carefully necropsied rural pigs and 191 slaughtered pigs from rural communities in which the presence of the Taenia solium metacestode was assessed by tongue dissection. Sera were analyzed by ELISA to detect antibodies against T. solium antigens and to detect parasite antigens. Most sera from the necropsied rural pigs were also evaluated by the Western blot method. Antigen and antibody ELISA detection assays showed high sensitivity and specificity when applied to sera from pigs reared in commercial farms. In contrast, all methods (Ag-ELISA, Ab-ELISA assays, EITB and tongue inspection) showed lower sensitivity and specificity when applied to the generally lightly infected rurally reared pigs. The probability distribution of cysts in carcasses were also determined. These results emphasize the difficulties in detecting cysticercosis in rural pigs with low levels of cyst burdens.

A protective role for IL-6 during early infection with Toxoplasma gondii.

IL-6 deficient mice were found to be significantly more susceptible to peroral infection with Toxoplasma gondii than their wild-type counterparts as measured by survival, brain cyst burdens and brain pathology at 28 days post infection. The physical manifestations of disease, such as weight loss, were not observed in IL-6 deficient animals until at least seven days later than such changes occurred in wildtype mice. During this early stage of infection IL-6+/+ but not IL-6-/- mice mounted a peripheral blood neutrophilia. Furthermore, between 6-8 days post-infection there was a significant increase in plasma IFN-gamma levels in wild-type but not IL-6 deficient mice. Not until days 18-23 post-infection, concurrent with the majority of deaths in IL-6-/- mice, were plasma IFN-gamma levels substantially and significantly raised in IL-6-/- mice. At this time not only were these plasma IFN-gamma levels 20-fold higher than background but eight-fold greater than peak (6-8 days post-infection) IFN-gamma levels in IL-6+/+ mice. IFN-gamma dependent parasite specific IgG2a levels were also significantly higher in IL-6-/- mice over this period and thereafter. Overall the evidence suggests that in the absence of IL-6 mice are unable to initiate a rapid proinflammatory response against T. gondii, which allows increased parasite growth. Increased mortality in IL-6-/- mice may be directly due to this increased parasite burden and the excessive inflammatory response this induces three weeks post-infection.