Dynamics of transcription of immunomodulatory genes in endothelial cells infected with different coccidian parasites.

Sporozoites of Eimeria bovis and tachyzoites of Neospora caninum and Toxoplasma gondii are able to invade and to replicate in endothelial cells. Here we report on responses of bovine umbilical vein endothelial cells (BUVEC) in vitro to these coccidial infections by determining mRNA levels of the CXC chemokines GRO-alpha, IL-8 and IP-10, the CC chemokines MCP-1 and RANTES and of GM-CSF, COX-2 and iNOS relative to the level of housekeeping gene (GAPDH) transcription. T. gondii and N. caninum tachyzoites caused profound transcriptional upregulation of all genes in question. In general, upregulation started 2-4 h p.i. and maximum transcript levels were observed 4 h p.i. GRO-alpha and IL-8 gene transcription had decreased to almost control levels by 12 h p.i.; in the case of the other chemokines enhanced transcript levels persisted longer or showed a biphasic time-course. A similar time-course to CC chemokines was observed for GM-CSF mRNA, whilst COX-2 gene transcript peaks were detected at 2-4 h p.i. and 48-72 h p.i. iNOS mRNA levels increased from 4 to 48 h p.i. In contrast, E. bovis sporozoites failed to induce the transcription of CXC chemokine genes and of COX-2, and only caused moderate transcription upregulation of the other genes considered. In conclusion, infections of BUVEC with these coccidian parasites result in host cell activation associated with enhanced transcription of genes encoding for proinflammatory and immunomodulatory molecules, which are important for innate immune reactions and the transition to adaptive immunity. Differences between E. bovis versus T. gondii and N. caninum may illustrate a particular evasion strategy of E. bovis sporozoites, which is related to their need to persist in the host cell for a long period of time and to the avoidance of inflammatory process-induction.

The route of immunization of gerbils with live Besnoitia besnoiti as a factor in protection against lethal challenge.

Gerbils (Meriones tristrami) were infected subcutaneously or intraperitoneally with live culture-grown Besnoitia besnoiti at doses ranging from 10(1) to 10(7) endozoites. All animals injected subcutaneously survived the infection and were refractory to a lethal challenge dose of 10(7) endozoites given intraperitoneally 6 weeks later. By contrast, gerbils surviving primary intraperitoneal inoculation with 10(2) to 10(6) endozoites showed a variable survival rate to challenge. All gerbils developed antibodies to B. besnoiti regardless of the route of inoculation, except for those given 10(1) endozoites intraperitoneally. There was no statistical difference between the immunofluorescent antibody titres developed in groups vaccinated subcutaneously or intraperitoneally (P = 0.556).

Specificity of the carbon immunoassay (CIA) test for the diagnosis of Toxoplasma infections.

A rapid and low cost procedure, the carbon immunoassay (CIA) test, was evaluated for the diagnosis of Toxoplasma gondii infections. Using a closely related parasite (Besnoitia jellisoni) as antigen, and homologous or heterologous immune sera, it was demonstrated by light and electron microscopy that CIA is a very reliable and specific test. As it is neither expensive nor time-consuming, it can be recommended for general and routine laboratory use.

Monoclonal antibodies raised against coccidia and malarial parasites recognize antigenic epitopes found in lankesterellid and adeleorin parasites.

Three murine monoclonal antibodies (MAbs) raised against poultry coccidia or murine malarial parasites were tested for cross-reactivity with 2 sporozoan parasites with different life histories and hosts: Lankesterella minima (Eimeriorina), an intraerythrocytic parasite of frogs that is transmitted by leeches; and Heptazoon catesbianae (Adeleinorina) that infects the red blood cells of frogs and is transmitted by mosquitoes. MAb 1209 recognized both refractile bodies of sporozoites of L. minima, using the indirect fluorescent antibody (IFA) technique and immunoelectron microscopy, and recognized antigens with relative rates of migration (M(r)) of 17, 23, 26, 43, and 48 kDa on a chemiluminescent western blot of L. minima sporozoite antigens. MAbs C(3)4F1 and E12 demonstrated spotty cytoplasmic staining and labeling of the anterior pellicle of L. minima sporozoites, respectively. Gamonts of H. catesbianae labeled with only MAb E12, using IFA. These gamonts exhibited staining similar to that observed with the L. minima sporozoites. The presence of the cross-reactive epitopes recognized by these MAbs in the same conserved locations suggests that these antigens are homologous.

Antigenic similarity between Hammondia hammondi and Toxoplasma gondii tachyzoites.

Hammondia hammondi is an obligate heteroxenous intestinal coccidian of cats, sharing many characteristics with Toxoplasma gondii. The tachyzoite stage antigens of T. gondii and H. hammondi were studied by immunofluorescence assays (IFA) and western blotting (WB) techniques to demonstrate antigenic similarities. Five monoclonal antibodies (MAbs), anti-T. gondii antigens, P22, P23, P30, P35, and P43, and mice polyclonal anti-H. hammondi serum were investigated. Antigens of H. hammondi were recognized by anti-P30 MAb both in IFA and in WB and by anti-P22 and anti-P35 MAbs only in IFA. Polyclonal anti-H. hammondi serum revealed many common antigens between the 2 parasites (30, 32, 35, 66, and 90 kDa). The differences of host parasite relationship between these 2 coccidians lead us to suggest that many of these antigens with similar molecular weights are not the same, but homologous, molecules or that they are not the only factors involved in these differences.

Temperature modulation of the response of Ig-positive cells to Goussia carpelli (Protozoa: Apicomplexa) infections in carp, Cyprinus Carpio L.

The influence of temperature on the kinetics of immunoglobulin-positive (Ig+) leukocytes in cell suspensions prepared from the pronephros and the intestine of common carp Cyprinus carpio during the development of Goussia carpelli, a gut-dwelling coccidian parasite, was examined. The development of the parasite was temperature dependent. At 20 C, oocysts were formed 2-3 wk postexposure (PB), at 15 C for 3-4 wk PE, and at 12 C for 5-6 wk PE. At all 3 temperatures, changes of relative numbers of Ig+ cells were observed in cell suspensions. During merogony and gamogony, the proportion of Ig+ cells increased, peaked during oocyst formation of the parasite, and then remained elevated. Serum collected from infected carp 8-20 days PE contained immunoglobulins binding to G. carpelli merozoites. The development of the parasite and the increase of Ig+ cells in intestine and pronephros of infected carp were temperature dependent. The peak level of Ig+ cells appeared not to be influenced by temperature. These findings indicate that even at low temperatures local and systemic immune responses are induced in carp with enteritic parasite infection.

Besnoitia sp. (Protozoa:Toxoplasmatinae) from Akodon montensis (Rodentia:Cricetidae) in Santa Catarina State, Brazil.

Tissue cysts of Besnoitia sp. were found in muscles and several organs from a naturally infected Akodon montensis captured in the rural area of the municipality of Timbó, Santa Catarina State, in southern Brazil. Indirect fluorescence and enzyme-linked immunosorbent assays carried out with sera from mice chronically infected with Toxoplasma gondii and Besnoitia sp. showed, as expected, a stronger reaction against homologous than heterologous antigens. No cross-protection was observed in mice immunized with T. gondii when challenged with Besnoitia sp. This is the first description of a natural infection of A. montensis by parasites of the genus Besnoitia sp. in Brazil.

Hammondia hammondi organelle proteins are recognized by monoclonal antibodies directed against organelles of Toxoplasma gondii.

Hammondia hammondi and Toxoplasma gondii, 2 closely related coccidia of cats, are known to share many antigenic molecules as shown by serologic cross reactivity. Monoclonal antibodies (MAbs) directed against the internal organelles of Toxoplasma gondii were tested by immunofluorescence assay and immunoelectron microscopy on the tachyzoites of H. hammondi. The MAbs anti-apex, anti-dense granules, anti-micronemes, and anti-rhoptries recognized, although weakly, the corresponding antigens on H. hammondi. This finding demonstrates that organelles of the 2 parasites are not only morphologically, but also antigenically, similar.

Studies on serological cross-reaction of Neospora caninum with Toxoplasma gondii and Hammondia heydorni.

The enzyme-linked immunosorbent assay (ELISA) was used to examine cross-reactivity of Neospora caninum with Toxoplasma gondii and Hammondia heydorni. Anti-T. gondii mouse and cat sera cross-reacted with N. caninum soluble antigen (NLA), but not with the recombinant surface antigen (NcSRS2). Anti-H. heydorni dog sera showed no cross-reactivity with either the NLA antigen or the NcSRS2. Lack of cross-reactivity between anti-H. heydorni sera and N. caninum antigens, and the cross-reactivity of anti-T. gondii sera with the NLA suggest that N. caninum has common antigens to T. gondii except for NcSRS2 based on serology. In light of several studies suggesting a closer relationship between N. caninum and H. heydorni than with T gondii, examination of serological cross-reactivity with N. caninum may be necessary to further classify the parasites in addition to molecular and morphological studies and clarification of the life cycle.

Bovine besnoitiosis: transfer of colostral antibodies with observations possibly relating to natural transmission of the infection.

Colostral antibodies to B. besnoiti were detected by immunofluorescence in four calves born to two Besnoitia-infected dams, with titres ranging from 1:64 to 1:1024. A specific antibody titre of 1:1024 was found in colostrum collected from one of the dams. Two of the newborn calves, when sampled immediately after birth, were serologically negative to B. besnoiti, but became positive on the next day. In all the calves, antibodies were detectable up to the age of 4 months. Observations concerning passive transfer of antibodies from Besnoitia-infected dams to offspring, and transmission of the infection among infected and non-infected closely kept cows, are discussed.

Activity of a monoclonal antibody against Besnoitia besnoiti endozoites.

Besnoitia besnoiti multiplication in vitro was inhibited by a specific organelle complex-directed monoclonal antibody (MoAb) raised against the endozoite stage. Multiplication rates in antibody-treated cultures were lower at parasite/cell ratios of 1:1, 1:2 and 1:10, than in untreated cultures, after 4 or 8 d of incubation. At 1:100 ratio, there was no difference between test and control cultures irrespective of the incubation period. In in vivo experiments, the anti-B besnoiti MoAb had no neutralizing effect on the infectivity of endozoites. Inoculation of gerbils with antibody-preincubated endozoites, followed by treatment with these MoAb through 5 successive days ultimately failed to prevent death in any of them. In Western blots the specific anti-B besnoiti MoAb of the IgG1 subclass recognized a 70 kDa endozoite protein in the cytosolic and the insoluble membrane fraction of endozoites.

Cross-reactivity of human Toxoplasma-specific T cells: implications for development of a potential immunotherapeutic or vaccine.

Previous reports from this laboratory have demonstrated that human CD4+ Toxoplasma-specific cytotoxic T cell (CTL) clones generated by stimulation of peripheral blood mononuclear cells with Toxoplasma RH strain antigens also recognized target cells expressing C strain antigens. To extend these observations, additional Toxoplasma isolates were studied. A simple system for assessment of cytotoxicity using T cell lines rather than cloned CTL was used. Stimulation of human peripheral blood mononuclear cells with Toxoplasma RH strain antigens elicited cytotoxic T cell lines specific for target cells expressing antigens derived from many other Toxoplasma strains. Cell lines produced by stimulation with antigens derived from the related, nonpathogenic coccidian Besnoitia jellesoni were also cytotoxic for target cells expressing Toxoplasma antigens. Proliferative responses to many Toxoplasma isolates and to the Toxoplasma p30 protein were also noted.

Toxoplasma gondii: a monoclonal antibody that inhibits intracellular replication.

During its intracellular life cycle within the infected host cell, Toxoplasma gondii is able to undergo rapid asexual replication. Neither the mechanism by which the parasite initiates this process nor the requirements for maintaining it are understood. We produced a monoclonal antibody, 1B8, that identifies a parasite antigen of approximate M(r) 97 kDa as determined by SDS-PAGE. The epitope recognized by mAb 1B8 appears as a collection of vesicular structures scattered throughout the cell cytoplasm. When RH strain parasites are incubated with mAb 1B8 in the absence of serum complement, parasite growth is inhibited by > 90% as determined by radioisotope incorporation. Both attachment and invasion assays show that neither of these parasite-host cell interactions are inhibited by the mAb. However, a marked reduction in the number of intracellular rosettes was observed following mAb treatment of the parasites. Viable extracellular parasites are able to endocytose mAb 1B8. Once within the parasite cytosol the antibody recognizes the vesicular structures similar to those observed with fixed parasites. Immunofluorescence assays with Besnoitia jellisoni and Plasmodium falciparum show that the epitope recognized by mAb 1B8 is conserved among Coccidiae but not the kinetoplastid Leishmania.

Invasion of different cell types by sporozoites of Eimeria species and effects of monoclonal antibody 1209-C2 on invasion of cells by sporozoites of several apicomplexan parasites.

Sporozoites of avian Eimeria species differed markedly in their ability to invade cells in vitro. Invasion by E. tenella and E. adenoeides was significantly greater in baby hamster kidney (BHK) and chicken cecal cell (CC) cultures than in primary chicken (PCK) or turkey kidney (PTK) cell cultures. Moreover, invasion of BHK cell cultures by E. adenoeides was significantly greater than that of other Eimeria species, and invasion by E. acervulina sporozoites was significantly lower. Monoclonal antibody 1209-C2 (MAb 1209-C2) reacted by immunofluorescent labeling (IFA) with refractile bodies of sporozoites of 5 species of Eimeria and Caryospora bigenetica, but not with sporozoites of Toxoplasma gondii, Hammondia hammondi, or Cryptosporidium parvum, which have no refractile bodies. The MAb also cross-reacted with formalin-fixed BHK, CC, turkey cecal (TC) cells, and PTK. Pretreatment of BHK cells with MAb 1209-C2 significantly reduced invasion of the cells by sporozoites of E. tenella, E. acervulina, E. meleagrimitis, and C. bigenetica, but did not alter invasion by T. gondii, C. parvum, or H. hammondia. Apparently, reactivity of MAB 1209-C2 with the sporozoites was required for inhibition of invasion despite the fact that the inhibition resulted from pre-treatment of the host cell. Conversely, although MAb 1209-C2 also reacted moderately with PTK and TC cells, pre-treatment of these cell cultures with the MAb did not inhibit invasion by either MAB 1209-C2-reactive or -nonreactive parasites. Collectively, the data indicated that refractile body antigens of sporozoites of Eimeria and Caryospora, which are recognized by MAb 1209-C2, may function in cellular invasion, but also suggest that cellular invasion is probably not mediated by interactions between the conserved epitopes in sporozoites and cultured host cells that are recognized by the MAb.

Differentiating between Besnoitia besnoiti from cattle and Sarcocystis hoarensis from rodents.

To provide a biological basis for studies designed to establish the mode of transmission of the veterinary pathogen Besnoitia besnoiti, we compared salient features of this pathogen in cattle with those of Sarcocystis hoarensis in rodents. The cysts and cystozoites of these organisms can readily be distinguished morphologically. In contrast to S. hoarensis, which is well adapted to rodents, B. besnoiti fails to mature in jirds or mice and generally is lethal in jirds. Serological reagents discriminately detect these pathogens. B. besnoiti, therefore, can unambiguously be differentiated from S. hoarensis either by morphological or serological methods or on the basis of experimental comparisons of virulence in laboratory rodents.