Mucosal immunity in Toxoplasma gondii infection.

Toxoplasma gondii is an intracellular parasite that frequently infects a large spectrum of warm-blooded animals. This parasite induces abortion and establishes both chronic and silent infections, particularly in the brain. Parasite penetration into the host activates a strong anti-parasite immune response. In the present paper, we will discuss the interplay between innate and adaptive immunity that occurs within the infected intestine to clear the parasite and to maintain intestinal homeostasis despite the exacerbation of an inflammatory immune response.

Release of organic matter in a discontinuously chlorinated drinking water network.

The effects of discontinuous chlorination on the characteristics of the water in a pilot drinking water distribution network were investigated. The release or consumption of organic matter (as dissolved organic carbon, DOC) following chlorination and non-chlorination periods were estimated, as were changes in bacterial cell production. In each unchlorinated network 0.3 mg DOCl(-1) was consumed and the average cell production was approximately 1.3 x 10(5) cells ml(-1). In discontinously chlorinated networks (chlorine treatment: 3.3 mg Cl2l(-1), chlorine residual: 0.1 mg Cl2l(-1)) the DOC release (DOCout-DOCin) was between 0.1 and 0.2 mg Cl(-1). Biomass production (cells(out)-cells(in)) during this chlorination period was lower (approximately 2 x 10(4) cells ml(-1)). The delay before DOC was released in chlorinated networks appeared to be less than 24 h, which corresponds to one hydraulic residence time. Likewise, when chlorination was stopped, 24 h or less were required before an efficient DOC removal was resumed. When chlorination was prolonged the observed release of DOC was progressively reduced from 0.2 mg l(-1) to zero, thus after 6 weeks of continuous chlorination the DOCin was equivalent to the DOCout.

Dynamics and responsiveness of T-lymphocytes in secondary lymphoid organs of rabbits developing immunity to Eimeria intestinalis.

Primary infection with Eimeria intestinalis confers very effective immunity against further infections in rabbits. This study was designed to determine the onset of the immune response in primary-infected rabbits and to characterise the immune status of protected rabbits. Variations in kinetics of CD4+ and CD8+ T-cell subpopulations were followed after primary infection at the intestinal sites of penetration (duodenum) and development (ileum), in mesenteric lymph nodes (MLN) and in the spleen. The response against the parasite was measured by specific lymphocyte proliferation in the spleen and MLN and by determining specific IgG titres in serum. The mucosal immune response was strong after primary infection and was characterised by (i) transient increase in the percentages of intestinal CD4+ lymphocytes and MLN CD8+ lymphocytes 14 days PI and (ii) strong increase in the percentages of intestinal CD8+ lymphocytes from 14 days PI persisting throughout further infections. Extensive infiltration of the lamina propria with CD8+ lymphocytes was observed 14 days PI. The specific proliferative response started between 7 and 14 days PI in MLN but remained undetectable in spleens for up to 21 days, in contrast to "immunised" rabbits. The fact that systemic immune responses were low after primary infection, in contrast to indicators of mucosal immune responsiveness, suggests that protection of rabbits against E. intestinalis infection is due to an effective mucosal immune response, and that systemic responses that increase after successive infections are only reflections of repeated encounters with parasite antigens.

Relationship between coliform culturability and organic matter in low nutritive waters.

The objective of this work was to investigate the behaviour of coliform bacteria in specific low nutritive waters conveying organic fractions from different origin of which an unknown part is likely to pass through the treatment barrier. For this purpose, we studied the growth (microscopic counting) and the culturability (count on nutritive medium) of ten coliform bacteria species as a function of the amount of organic matter in a river water collected after a period of heavy rain and in an algal bloom water. Assays were carried out in the presence of autochthonous heterotrophic bacteria from the Nancy (France) drinking water, with variable concentrations of dissolved organic carbon (DOC) representative of drinking waters (0.5-1.5 mg l(-1) for diluted river water samples and 1.3-2.5 mg l(-1) for diluted algal bloom water samples). Bacterial growth was measured in the two types of water, regardless of the initial concentration of DOC. We found that coliform bacteria lost their culturability in both sample series, and that the lower the initial DOC concentration the more rapidly the culturability was lost. The quantity of DOC consumed by the bacteria in the two water types (0.03-0.13 mg l(-1) in river water and 0.77-1.29 mg l(-1) in algal bloom water) and the resulting consequences on bacterial behaviour suggested that bloom water contains algal organic compounds that are antagonistic to the growth and/or the culturability of coliform bacteria. Organic matter thresholds, beyond which coliform bacteria are unlikely to keep their culturability, have not been determined experimentally. Indeed, at the end of the assays some culturable coliform bacteria were systematically detected in both types of water. Enterobacter cloacae was the predominant species. Thus, during these adverse events the probability of coliform occurrence can be considered as high in treated water.

Anti-SAG1 peptide antibodies inhibit the penetration of Toxoplasma gondii tachyzoites into enterocyte cell lines.

The initial attachment of Toxoplasma tachyzoites to the target host cell is an important event in the life-cycle of the parasite and a critical stage in infection. Previous studies have shown that polyclonal antibodies directed against the major surface antigen of Toxoplasma gondii (SAG1) inhibit the infection of enterocyte cell lines. Here, we demonstrate that antibodies raised against a central peptide (V41T) of SAG1 and the SAGI protein itself are able to inhibit the infection of various cell lines by the tachyzoites. Antibodies directed against SAG1 peptides were used to define a site on the SAGI antigen that interacts with the host cell. The epitope carried by V41T was identified on the tachyzoite surface by immunofluorescence. The peptide sequence seems to be conserved in all the members of the SAGI Related Sequence family (SRS). Using undifferentiated and differentiated Caco-2 cells, we found that tachyzoites enter preferentially via the basolateral side of the cell. These findings highlight the role of the SRS family members in the mediation of host cell invasion.

Monitoring and modeling of disinfection by-products (DBPs).

In the United States, the newly promulgated disinfectant/disinfection by-product (D/DBP) regulations force water treatment utilities to be more concerned with finished and distributed water qualities. In this study, monitoring of DBP formation was conducted from three French water treatment plants trying to assess DBP variations through time and space. Compared to the in-plant total trihalomethanes (TTHM) levels, TTHM levels in the distribution system increased from less than 150% to more than 300%. Significant variations for TTHM and bromate (BrO3-) levels throughout the seasons were also observed; generally higher levels in the summer and lower levels in the winter. Combining chemical DBP models (empirical power functional models) and hydraulic simulations, DBPs including TTHM and BrO3- were successfully simulated from the full-scale monitoring data, indicating that empirical DBP model can be a potential tool to access DBP formation in actual plants. This study also provides the protocols to assess DBP simulations in the water treatment systems.

Murine ileitis after intracellular parasite infection is controlled by TGF-beta-producing intraepithelial lymphocytes.

BACKGROUND & AIMS: Acute inflammatory ileitis occurs in susceptible (C57BL/6) mice after oral infection with Toxoplasma gondii. Overproduction of interferon (IFN)-gamma and synthesis of nitric oxide mediate the inflammation. We evaluated the role of transforming growth factor (TGF)-beta produced by intraepithelial lymphocytes (IELs) in this process. METHODS: We analyzed the histologic and immunologic consequences of adoptive transfer of antigen-primed IELs into susceptible mice treated with anti-TGF-beta before oral challenge with T. gondii cysts. An in vitro coculture of enterocytes and IELs assessed the production of chemokines and cytokines in the presence of anti-TGF-beta. RESULTS: Antigen-primed IELs prevent acute ileitis in susceptible mice that is reversed with anti-TGF-beta. Resistant mice (CBA/J) develop ileitis after treatment with anti-TGF-beta. Antigen-primed IELs can induce systemic immunosuppression as measured by depressed IFN-gamma production. In vitro, primed IELs reduce the production of inflammatory chemokines by infected enterocytes and IFN-gamma by splenocytes. CONCLUSIONS: Regulation of the ileal inflammatory process resulting from T. gondii is dependent on TGF-beta-producing IELs. The IELs are an essential component in gut homeostasis after oral infection with this parasite.

Effect of adding phosphate to drinking water on bacterial growth in slightly and highly corroded pipes.

The effect of phosphate addition in drinking water was tested under static conditions as batch tests and under dynamic conditions using continuously fed reactors. Phosphate supplements in batch tests from 0.1 to 2 mg P-PO4 L(-1) did not show any relationship between bacterial growth and phosphate concentration. Dynamic tests in slightly corroded reactor (stainless steel) treated at 1 mg P-PO4 L(-1) showed only a moderate improvement in the growth of microorganisms. On the contrary, phosphate treatment applied to the highly corroded reactor (unlined cast iron) led to an immediate, drastic drop in iron oxide release and bacterial production. Phosphate uptake by the reactor wall was less than 14% with the stainless-steel reactor and 70-90% with the corroded cast iron reactor. Moreover, about 5% of the phosphate associated to corroded iron pipe walls was released for 20 days after the end of treatment.

Intranasal immunization with SAG1 and nontoxic mutant heat-labile enterotoxins protects mice against Toxoplasma gondii.

Effective protection against intestinal pathogens requires both mucosal and systemic immune responses. Intranasal administration of antigens induces these responses but generally fails to trigger a strong protective immunity. Mucosal adjuvants can significantly enhance the immunogenicities of intranasally administered antigens. Cholera toxin (CT) and heat-labile enterotoxin (LT) are strong mucosal adjuvants with a variety of antigens. Moreover, the toxicities of CT and LT do not permit their use in humans. Two nontoxic mutant LTs, LTR72 and LTK63, were tested with Toxoplasma gondii SAG1 protein in intranasal vaccination of CBA/J mice. Vaccination with SAG1 plus LTR72 or LTK63 induced strong systemic (immunoglobulin G [IgG]) and mucosal (IgA) humoral responses. Splenocytes and mesenteric lymph node cells from mice immunized with LTR72 plus SAG1, but not those from mice immunized with LTK63 plus SAG1, responded to restimulation with a T. gondii lysate antigen in vitro. Gamma interferon and interleukin 2 (IL-2) production by splenocytes and IL-2 production by mesenteric lymph node cells were observed in vitro after antigen restimulation, underlying a Th1-like response. High-level protection as assessed by the decreased load of cerebral cysts after a challenge with the 76K strain of T. gondii was obtained in the group immunized with LTR72 plus SAG1 and LTK63 plus SAG1. They were as well protected as the mice immunized with the antigen plus native toxins. This is the first report showing protection against a parasite by using combinations of nontoxic mutant LTs and SAG1 antigen. These nontoxic mutant LTs are now attractive candidates for the development of mucosally delivered vaccines.

In vitro activity of the polyether ionophorous antibiotic monensin against the cyst form of Toxoplasma gondii.

Toxoplasma gondii. The experiments were conducted in vitro using 2 methods; cysts produced either in mice or in cell culture were exposed to monensin in vitro, and the infectivity of the parasites was then assessed in vivo or in vitro. The data obtained from these 2 systems of evaluation showed that monensin inhibits the infectivity and the viability of the bradyzoites. Its activity was time and concentration dependent. The first effects were observed at very low drug concentrations (i.e. 0.0001 microg/ml). Immunofluorescence and electron microscopy analysis showed significant cytological alterations of the monensin-treated bradyzoites: they were swollen, had a large number of vacuoles in their cytoplasm and were found lysed at higher concentrations in ionophore.

Induction of protective immunity against toxoplasmosis in mice by DNA immunization with a plasmid encoding Toxoplasma gondii GRA4 gene.

GRA4 is a dense granule protein of Toxoplasma gondii that is a candidate for vaccination against this parasite. We have inserted the entire coding sequence of GRA4 into an eukaryotic expression vector to determine whether DNA immunization can elicit protective immune response to T. gondii. Susceptible C57BL/6 mice were then vaccinated intramuscularly with GRA4 DNA and orally challenged with a lethal dose of 76 K T. gondii strain cysts. Immunization with pGRA4 resulted in a 62% survival of C57BL/6 infected mice. Mice immunized with GRA4 DNA developed high levels of serum anti-GRA4 immunoglobulin G antibodies as well as a cellular immune response, as assessed by splenocyte proliferation, in response to recombinant GRA4 protein restimulation in vitro. The cellular immune response was associated with IFN-gamma and IL-10 synthesis, suggesting a modulated Th1-type response. Splenocyte proliferation was strongly enhanced and protection slightly higher by inoculation with GRA4 DNA combined with a granulocyte-macrophage colony-stimulating factor expressing vector. This is the first report that demonstrates the establishment of a DNA vaccine-induced protective immunity against the acute phase of T. gondii infection.

Intranasal immunization with Toxoplasma gondii SAG1 induces protective cells into both NALT and GALT compartments.

Intranasal (i.n.) immunization with the SAG1 protein of Toxoplasma gondii plus cholera toxin (CT) provides protective immunity. The aim of this study was to analyze the cellular activation of several mucosal compartments after i.n. immunization. Cervical and mesenteric lymph node (CLN and MLN, respectively) lymphoid cell and intraepithelial lymphocyte (IEL) passive transfer experiments were performed with CBA/J mice immunized i.n. with SAG1 plus CT. CLN and MLN cells and IEL isolated 42 days after immunization conferred protective immunity on naive recipient mice challenged with strain 76K T. gondii, as assessed by the reduction in the number of brain cysts. There were proliferative specific responses in nose-associated lymphoid tissue and the CLN and MLN cells from mice immunized with SAG1 plus CT, but no cytokine was detectable. Thus, protective immunity is associated with a specific cellular response in the nasal and mesenteric compartments after i.n. immunization.

Nasal immunization of mice with human papillomavirus type 16 (HPV-16) virus-like particles or with the HPV-16 L1 gene elicits specific cytotoxic T lymphocytes in vaginal draining lymph nodes.

Human papillomavirus type 16 (HPV-16) infects the genital tract and is closely associated with the development of cervical cancer. HPV-16 initiates infection at the genital mucosal surface; thus, mucosal immune responses are likely to contribute to defense against HPV-16 infection. However, little information is available regarding the induction of immune responses in the genital tract mucosa. In this study, we evaluated the potential of intranasally administered papillomavirus vaccines to elicit both systemic and vaginal immune responses. HPV-16 virus-like particles (VLPs) produced by self-assembly of L1 protein and the HPV-16 L1 gene cloned into a mammalian expression vector were used as vaccines. Intranasally administered VLPs induced serum immunoglobulin G (IgG) and vaginal IgA secretory antibodies. Very weak serum IgG and vaginal IgA responses were found after DNA immunization. Both splenic and vaginal lymphocytes could be activated by intranasal immunization with VLPs and the HPV-16 L1 gene. Activated CD4(+) Th1-like T cells were shown to synthesize gamma interferon, and activated CD8(+) T cells were demonstrated to be cytotoxic.

Protection of kids against Cryptosporidium parvum infection after immunization of dams with CP15-DNA.

In this study the effectiveness of a DNA vaccine to confer protection against cryptosporidiosis, an enteric infection of lifestock and humans, was evaluated. A vaccination protocol using a recombinant plasmid encoding the 15 kDa surface sporozoite protein of Cryptosporidium parvum was developed in adult pregnant goats. The present study reports that nasal immunization of pregnant goats with CP15-DNA led to a transfer of immunity to offspring conferring protection against C. parvum infection. Kids from CP15-DNA-vaccinated dams shed significantly fewer oocysts and over a shorter period than did kids from unvaccinated goats. The low level of parasite development in protected kids did not affect their growth whereas unprotected kids grew much slowly. There was still a significant difference in the weights of protected and unprotected kids after complete recovery. Anti-CP15 antibodies were present in serum and colostrum from vaccinated goats. Nevertheless, the precise immune mechanism of protection has still to be determined. This vaccine should reduce the economic losses due to cryptosporidiosis in ruminants, specially in small ruminants (calves, lambs, kids). It has also the potential to reduce environmental contamination by reducing oocyst shedding.

Intraepithelial lymphocytes traffic to the intestine and enhance resistance to Toxoplasma gondii oral infection.

Toxoplasma gondii Ag-primed intraepithelial lymphocytes (IEL) from the mouse intestine have been shown to be protective against an lethal parasite challenge when adoptively transferred into recipient mice. In the present study, we observed that Ag-primed IEL traffic to the intestine of naive mice following i.v. administration. Primed and CD8beta+ IEL were the most efficient cells at homing to the host organ. In congenic mice, IEL migrated from intestine within several hours posttransfer. On Ag reexposure, the primed IEL return to the intestine where they enhance resistance as determined by reduction in the number of brain cysts. Treatment of recipient mice with anti-alpha4 and anti-alphaE Abs partially inhibited IEL intestinal homing. The Ab treatment dramatically impaired resistance to a subsequent oral infection. These finding indicate that lymphocyte homing is an important parameter in establishing long term immunity to recurrent infection with this parasite.

Interaction between Toxoplasma gondii and enterocyte.

Enterocyte is the first cell to be invaded by Toxoplasma gondii when ingested parasites are released from cysts or oocysts within the gastrointestinal tract. Our data showed that the transcytotic pathway of IgA could interfere with intracellular replication of T. gondii. On another hand, IFN-gamma could activate enterocyte and inhibit the parasite replication through an iron-dependent mechanism.

Cryptosporidium removal

El tratamiento del agua para la remocion de Cryptosporidium depende de diversos factores, como el area de la fuente del agua y el tipo de polucion presente en ella. Se analizan posibles tratamientos empleados en Europa, principalmente en Francia, para la eliminacion del parasito del agua. Se analizan ademas algunos parametros que permitan estimar el riesgo de infeccion por la presencia de oocistos

Gut-derived intraepithelial lymphocytes induce long term immunity against Toxoplasma gondii.

Intraepithelial lymphocytes (IEL) of the intestine represent an important barrier in the prevention of infection against orally acquired pathogens. Adoptive transfer of Ag-primed IEL into a naive host can protect against challenge. Using a murine model, we demonstrate in two genetically distinct mouse strains (C57BL/6 and CBA/J) that protective IEL can be isolated at specific times after oral infection with cysts containing bradyzoites. Adoptive transfer of IEL obtained from the intestine of infected mice at these specific times can provide long term protection, as determined by mortality and cyst number against challenge. The protective IEL appear to be CD8+, TCR-alpha/beta and are at least partially dependent upon the presence of TCR-gamma/delta T cells in the host. Endogenous production of the pivotal cytokine, IFN-gamma, is essential for host immunity. These findings demonstrate that gut-derived IEL represent a potentially important mechanism to provide long term immunity to the host.