Antigenic evaluation of a recombinant baculovirus-expressed Sarcocystis neurona SAG1 antigen

Sarcocystis neurona is the primary parasite associated with equine protozoal myeloencephalitis (EPM). This is a commonly diagnosed neurological disorder in the Americas that infects the central nervous system of horses. Current serologic assays utilize culture-derived parasites as antigen. This method requires large numbers of parasites to be grown in culture, which is labor intensive and time consuming. Also, a culture-derived whole-parasite preparation contains conserved antigens that could cross-react with antibodies against other Sarcocystis species and members of Sarcocystidae such as Neospora spp., Hammondia spp., and Toxoplasma gondii. Therefore, there is a need to develop an improved method for the detection of S. neurona-specific antibodies. The sera of infected horses react strongly to surface antigen 1 (SnSAG1), an approximately 29-kDa protein, in immunoblot analysis, suggesting that it is an immunodominant antigen. The SnSAG1 gene of S. neurona was cloned, and recombinant S. neurona SAG1 protein (rSnSAG1-Bac) was expressed with the use of a baculovirus system. By immunoblot analysis, the rSnSAG1-Bac antigen detected antibodies to S. neurona from naturally infected and experimentally inoculated equids, cats, rabbit, mice, and skunk. This is the first report of a baculovirus-expressed recombinant S. neurona antigen being used to detect anti-S. neurona antibodies in a variety of host species.     

Strains of Sarcocystis neurona exhibit differences in their surface antigens, including the absence of the major surface antigen SnSAG1

A gene family of surface antigens is expressed by merozoites of Sarcocystis neurona, the primary cause of equine protozoal myeloencephalitis (EPM). These surface proteins, designated SnSAGs, are immunodominant and therefore excellent candidates for development of EPM diagnostics or vaccines. Prior work had identified an EPM isolate lacking the major surface antigen SnSAG1, thus suggesting there may be some diversity in the SnSAGs expressed by different S. neurona isolates. Therefore, a bioinformatic, molecular and immunological study was conducted to assess conservation of the SnSAGs. Examination of an expressed sequence tag (EST) database revealed several notable SnSAG polymorphisms. In particular, the EST information implied that the EPM strain SN4 lacked the major surface antigen SnSAG1. The absence of this surface antigen from the SN4 strain was confirmed by both Western blot and Southern blot. To evaluate SnSAG polymorphisms in the S. neurona population, 14 strains were examined by Western blots using monospecific polyclonal antibodies against the four described SnSAGs. The results of these analyses demonstrated that SnSAG2, SnSAG3, and SnSAG4 are present in all 14 S. neurona strains tested, although some variance in SnSAG4 was observed. Importantly, SnSAG1 was not detected in seven of the strains, which included isolates from four cases of EPM and a case of fatal meningoencephalitis in a sea otter. Genetic analyses by PCR using gene-specific primers confirmed the absence of the SnSAG1 locus in six of these seven strains. Collectively, the data indicated that there is heterogeneity in the surface antigen composition of different S. neurona isolates, which is an important consideration for development of serological tests and prospective vaccines for EPM. Furthermore, the diversity reported herein likely extends to other phenotypes, such as strain virulence, and may have implications for the phylogeny of the various Sarcocystis spp. that undergo sexual stages of their life cycle in opossums.     

Detection of antibodies against Sarcocystis neurona, Neospora spp., and Toxoplasma gondii in horses from Costa Rica

Serum samples from 315 horses from Costa Rica, Central America, were examined for the presence of antibodies against Sarcocystis neurona, Neospora spp., and Toxoplasma gondii by using the surface antigen (SAG) SnSAG2 enzyme-linked immunosorbent assay (ELISA), the NhSAG1 ELISA, and the modified agglutination test, respectively. Anti- S. neurona antibodies were found in 42.2% of the horses by using the SnSAG2 ELISA. Anti- Neospora spp. antibodies were found in only 3.5% of the horses by using the NhSAG1 ELISA, and only 1 of these horses was confirmed seropositive by Western blot. Antibodies to T. gondii were found in 34.0% of the horses tested, which is higher than in previous reports from North and South America. The finding of anti- S. neurona antibodies in horses from geographical areas where Didelphis marsupialis has wide distribution suggests that D. marsupialis is a potential definitive host for this parasite and a source of infection for these horses.     

Horses experimentally infected with Sarcocystis neurona develop altered immune responses in vitro

Equine protozoal myeloencephalitis (EPM) due to Sarcocystis neurona infection is 1 of the most common neurologic diseases in horses in the United States. The mechanisms by which most horses resist disease, as well as the possible mechanisms by which the immune system may be suppressed in horses that develop EPM, are not known. Therefore, the objectives of this study were to determine whether horses experimentally infected with S. neurona developed suppressed immune responses. Thirteen horses that were negative for S. neurona antibodies in serum and cerebrospinal fluid (CSF) were randomly assigned to control (n = 5) or infected (n = 8) treatment groups. Neurologic exams and cerebrospinal fluid analyses were performed prior to, and following, S. neurona infection. Prior to, and at multiple time points following infection, immune parameters were determined. All 8 S. neurona-infected horses developed clinical signs consistent with EPM, and had S. neurona antibodies in the serum and CSF. Both infected and control horses had increased percentages (P < 0.05) of B cells at 28 days postinfection. Infected horses had significantly decreased (P < 0.05) proliferation responses as measured by thymidine incorporation to nonspecific mitogens phorbol myristate acetate (PMA) and ionomycin (I) as soon as 2 days postinfection.     

Are Sarcocystis neurona and Sarcocystis falcatula synonymous? A horse infection challenge

Equine protozoal myeloencephalitis (EPM) is a debilitating neurologic disease of the horse. The causative agent. Sarcocystis neurona, has been suggested to be synonymous with Sarcocystis falcatula, implying a role for birds as intermediate hosts. To test this hypothesis, opossums (Didelphis virginiana) were fed muscles containing S. falcatula sarcocysts from naturally infected brown-headed cowbirds (Molothrus ater). Ten horses were tested extensively to ensure no previous exposure to S. neurona and were quarantined for 14 days, and then 5 of the horses were each administered 10(6) S. falcatula sporocysts collected from laboratory opossums. Over a 12-wk period, 4 challenged horses remained clinically normal and all tests for S. neurona antibody and DNA in serum and cerebrospinal fluid were negative. Rechallenge of the 4 seronegative horses had identical results. Although 1 horse developed EPM, presence of S. neurona antibody prior to challenge strongly indicated that infection occurred before sporocyst administration. Viability of sporocysts was confirmed by observing excystation in equine bile in vitro and by successful infection of naive brown-headed cowbirds. These data suggest that S. falcatula and S. neurona are not synonymous. One defining distinction is the apparent inability of S. falcatula to infect horses, in contrast to S. neurona, which was named when cultured from equine spinal cord.     

Early migration of Sarcocystis neurona in ponies fed sporocysts

Sarcocystis neurona is the most important cause of equine protozoal myeloencephalitis (EPM), a neurologic disease of the horse. In the present work, the kinetics of S. neurona invasion is determined in the equine model. Six ponies were orally inoculated with 250 x 10(6) S. neurona sporocysts via nasogastric intubation and killed on days 1, 2, 3, 5, 7, and 9 postinoculation (PI). At necropsy, tissue samples were examined for S. neurona infection. The parasite was isolated from the mesenteric lymph nodes at 1, 2, and 7 days PI; the liver at 2, 5, and 7 days PI; and the lungs at 5, 7, and 9 days PI by bioassays in interferon gamma gene knock out mice (KO) and from cell culture. Microscopic lesions consistent with an EPM infection were observed in brain and spinal cord of ponies killed 7 and 9 days PI. Results suggest that S. neurona disseminates quickly in tissue of naive ponies.     

Prophylactic administration of ponazuril reduces clinical signs and delays seroconversion in horses challenged with Sarcocystis neurona

The ability of ponazuril to prevent or limit clinical signs of equine protozoal myeloencephalitis (EPM) after infection with Sarcocystis neurona was evaluated. Eighteen horses were assigned to 1 of 3 groups: no treatment, 2.5 mg/kg ponazuril, or 5.0 mg/kg ponazuril. Horses were administered ponazuril, once per day, beginning 7 days before infection (study day 0) and continuing for 28 days postinfection. On day 0, horses were stressed by transport and challenged with 1 million S. neurona sporocysts per horse. Sequential neurologic examinations were performed, and serum and cerebrospinal fluid were collected and assayed for antibodies to S. neurona. All horses in the control group developed neurologic signs, whereas only 71 and 40% of horses in the 2.5 and 5.0 mg/kg ponazuril groups, respectively, developed neurologic abnormalities. This was significant at P = 0.034 by using Fisher exact test. In addition, seroconversion was decreased in the 5.0 mg/kg group compared with the control horses (100 vs. 40%; P = 0.028). Horses with neurologic signs were killed, and a post-mortem examination was performed. Mild-to-moderate, multifocal signs of neuroinflammation were observed. These results confirm that treatment with ponazuril at 5.0 mg/kg minimizes, but does not eliminate, infection and clinical signs of EPM in horses.     

Preparation and properties of cloning inhibitory factor. II. Factors affecting its production and assay

Cloning Inhibition Factor (CIF), an activity present in PHA or antigen stimulated lymphocyte culture supernatants, inhibited the cloning of HeLa cells when diluted 1:9 in HeLa culture medium. CIF was not detectable at 8 hr, was maximal at 24–48 hr, and declined with longer periods of lymphocyte culture. CIF production increased with lymphocyte concentration up to 1–2 × 10⁶ lymphs/ml but plateaued at higher concentrations. At lower lymphocyte concentrations, more CIF activity was present when lymphocytes were cultured in 5% rather than 12% serum. PPD elicited similar CIF activity from either highly purified or unpurified lymphocytes. CIF activity was independent of HeLa medium serum concentration. It remained stable for 3–6 months at ?20 °C, but was inactivated by heating at 56 °C for 30 min. At a 1:9 dilution CIF was not cytocidal but produced cytopathic changes. CIF shares many properties with, and may be identical to, Proliferation Inhibitory Factor.     

Regulation of lymphocyte responses by human gangliosides. I. Characteristics of inhibitory effects and the induction of impaired activation

Gangliosides obtained from normal human brain were found to inhibit the in vitro activation of human lymphocytes by nonspecific mitogens and allogeneic cells at concentrations between 3 to 50 microgram/1.5 to 1.7 X 10(5) lymphocytes/0.2 ml culture. Ganglioside inhibition did not represent cytotoxic effects or altered lectin binding and was independent of the mitogen concentration. In addition to concentration, the degree of inhibition was dependent on the mode of presentation to lymphocytes, since gangliosides incorporated within liposomal membranes displayed a synergistic inhibitory effect greater than predicted from the cultures receiving either gangliosides or liposomes alone. In binding experiments, radiolabeled ganglioside GM1 became associated with human lymphocytes within 10 min. However, approximately 72 hr pre-exposure of human lymphocytes to gangliosides was required to induce impaired lymphocyte responses to mitogens and allogeneic cells. Thus, concentrations of human gangliosides equivalent to the levels occurring in the sera of patients with certain malignancies are capable of actively inhibiting lymphocyte stimulation in addition to inducing impaired lymphocyte responses.     

Depletion of natural killer cells does not result in neurologic disease due to Sarcocystis neurona in mice with severe combined immunodeficiency

Sarcocystis neurona is an apicomplexan parasite that is the primary etiologic agent of equine protozoal myeloencephalitis in horses. Protective immune responses in horses have not been determined, but interferon-gamma (IFN-gamma) is considered critical for protection from neurologic disease in mice. The role of adaptive and innate immune responses in control of parasites was explored by infecting BALB/c, IFN-gamma knockout (GKO), and severe combined immune deficient (SCID) mice with S. neurona (10(4) sporocysts/mouse). Immune competent BALB/c mice eliminated parasites within 30 days, with no sign of neurologic disease, whereas GKO mice developed fulminant neurologic disease. In contrast, SCID mice remained healthy throughout the experimental period despite the persistence of parasite at low levels in some mice. Treatment with anti-IFN-gamma antibody resulted in neurologic disease in infected SCID mice. Although SCID mice lack adaptive immune responses, they have natural killer (NK) cells capable of producing significant quantities of IFN-gamma. Therefore, SCID mice were infected with sporocysts of S. neurona and treated with anti-asialo GM1. Depletion of NK cells, confirmed by flow cytometry, did not result in neurologic disease in SCID mice. These results indicate that IFN-gamma mediates protection from neurologic disease in SCID mice. Protective levels of IFN-gamma may originate from a low number of nondepleted NK cells or from a non-T cell, non-NK cell population.     

A series of murine interleukin molecules which stimulate both murine and human lymphocytes: I. Production by Phytolacca americana (pokeweed) lectin 2 (Pa-2)-stimulated thymus and thymus-derived cells

Cell-free supernatant fluid, from cultures of Phytolacca americana (pokeweed) lectin 2 (Pa-2)-pulsed murine spleen or thymus cells, contains factors which induce cultured lymphocytes to differentiate into IgM-secreting cells (assayed by a reverse plaque technique) and to proliferate (measured by the incorporation of tritiated thymidine) without the addition of mitogen. The factors in this supernatant fluid responsible for these activities have been designated as lymphocyte stimulating factors (LSF). LSF showed no genetic restrictions related to the major histocompatability complex; LSF made in one strain of mice worked in other strains. Indeed, LSF is not restricted by species barriers; human peripheral blood mononuclear cells were also stimulated by murine LSF to proliferate and differentiate into immunoglobulin-secreting cells without further addition of antigen or mitogen. Maximum production of LSF was achieved within 12 hr of culture and was independent of cell division. In contrast to TRF, no further production of LSF was detectable after 24 hr of culture. Unlike T-cell growth factor, this material stimulated increased mitosis of thymic, T, and B lymphocytes without the addition of mitogen or antigen. LSF also stimulated polyclonal B-cell differentiation into IgM-secreting cells. Maximal numbers of immunoglobulin-secreting cells were generated when LSF was added at the initiation of the culture. Indeed, unlike TRF, LSF needed to be present only during the first 6 hr of culture to achieve maximum stimulation, and did not require the presence of antigen. The production of LSF by a T-cell population in the spleen was shown by two independent methods. Spleen cells treated with anti-Thy 1 plus complement failed to produce detectable levels of LSF. On the other hand, purified populations of surface immunoglobulin-negative spleen cells produced LSF. Furthermore, the subset of thymocytes responsible for LSF production was the small population (approximately 10%) of cells in the thymus, which are not agglutinated by peanut agglutinin.     

Equine protozoal myelitis in Panamanian horses and isolation of Sarcocystis neurona.

Schizonts of Sarcocystis neurona were identified microscopically in hematoxylin-eosin-stained spinal cord sections from 2 native Panamanian horses that exhibited clinical signs of equine protozoal myelitis (EPM). Spinal cord homogenate from a third Panamanian horse with EPM was inoculated onto monolayers of cultured bovine monocytes (M617). Intracytoplasmic schizonts containing merozoites arranged in rosette forms surrounding a central residual body first were observed 13 wk postinoculation. Parasites divided by endopolygeny and lacked rhoptries. Schizonts from each horse reacted with Sarcocystis cruzi antiserum in an immunohistochemical test.     

Prevalence of agglutinating antibodies to Sarcocystis neurona in raccoons (Procyon lotor) from an urban area of Virginia

Equine protozoal myeloencephalitis is the most important protozoan disease of horses in North America and is usually caused by Sarcocystis neurona. Natural and experimentally induced cases of encephalitis caused by S. neurona have been reported in raccoons (Procyon lotor) and raccoons are an intermediate host for this parasite. A 3-yr-long serological survey was conducted to determine the prevalence of agglutinating antibodies to S. neurona in raccoons collected from Fairfax County, Virginia, a suburban-urban area outside Washington, D.C. Samples from 469 raccoons were examined, and agglutinating antibodies (> or = 1:50 dilution) were found in 433 (92.3%) of the raccoons. This study indicates that exposure to S. neurona is high in this metropolitan area.     

Prevalence of antibodies to Neospora caninum, Sarcocystis neurona, and Toxoplasma gondii in wild horses from central Wyoming

Sarcocystis neurona, Neospora caninum, N. hughesi, and Toxoplasma gondii are 4 related coccidians considered to be associated with encephalomyelitis in horses. The source of infection for N. hughesi is unknown, whereas opossums, dogs, and cats are the definitive hosts for S. neurona, N. caninum, and T. gondii, respectively. Seroprevalence of these coccidians in 276 wild horses from central Wyoming outside the known range of the opossum (Didelphis virginiana) was determined. Antibodies to T. gondii were found only in 1 of 276 horses tested with the modified agglutination test using 1:25, 1:50, and 1:500 dilutions. Antibodies to N. caninum were found in 86 (31.1%) of the 276 horses tested with the Neospora agglutination test--the titers were 1:25 in 38 horses, 1:50 in 15, 1:100 in 9, 1:200 in 8, 1:400 in 4, 1:800 in 2, 1:1,600 in 2, 1:3,200 in 2, and 1:12,800 in 1. Antibodies to S. neurona were assessed with the serum immunoblot; of 276 horses tested, 18 had antibodies considered specific for S. neurona. Antibodies to S. neurona also were assessed with the S. neurona direct agglutination test (SAT). Thirty-nine of 265 horses tested had SAT antibodies--in titers of 1:50 in 26 horses and 1:100 in 13. The presence of S. neurona antibodies in horses in central Wyoming suggests that either there is cross-reactivity between S. neurona and some other infection or a definitive host other than opossum is the source of infection. In a retrospective study, S. neurona antibodies were not found by immunoblot in the sera of 243 horses from western Canada outside the range of D. virginiana.     

Novel mechanism for Trypanosoma cruzi-induced suppression of human lymphocytes. Inhibition of IL-2 receptor expression

Co-culture of blood forms of Trypanosoma cruzi, the causative agent of Chagas' disease, with human PBMC impaired the capacity of T lymphocytes to express surface receptors for IL-2. This effect was evidenced by marked reductions in both the proportion of Tac+ cells and the density of Tac Ag on the surface of the positive cells, determined by flow cytometry. The extent of the inhibition increased with parasite concentration. Under optimal or suboptimal conditions of stimulation with either PHA or monoclonal anti-CD3, specific for an epitope of the T3-Ti human T cell Ag receptor complex, the presence of T. cruzi curtailed the capacity of T lymphocytes to proliferate and express Il-2R but did not affect IL-2 production. Furthermore, the addition of exogenous IL-2 did not restore the responsiveness of suppressed human lymphocytes but did when mouse lymphocytes were used instead. Therefore, unlike mouse lymphocytes, human lymphocyte suppression by T. cruzi did not involve deficient IL-2 production and was accompanied by impaired IL-2 utilization. Co-culture of human monocytes/macrophages with suppressive concentrations of T. cruzi increased IL-1 production, and the parasite did not decrease IL-1 secretion stimulated by a bacterial LPS. Therefore, the suppression of IL-2R expression and lymphoproliferation is not likely to have been an indirect consequence of insufficient IL-1 production due to infection of monocytes or macrophages. We have shown that suppression of human lymphocyte proliferation by T. cruzi is not caused by nutrient consumption, absorption of IL-2, lymphocyte killing, or mitogen removal by the parasite. Therefore, these results uncover a novel suppressive mechanism induced by T. cruzi, involving inhibited expression of IL-2R after lymphocyte activation and rendering T cells unable to receive the IL-2 signal required for continuation of their cell cycle and mounting effective immune responses.     

T lymphocyte development and maturation in horses

Monoclonal antibodies specific for equine T lymphocyte subpopulations were produced and procedures for the continuous culture of equine lymphocytes were developed. These reagents and procedures were used to analyse the appearance, maturation and functions of T lymphocytes in normal horses and in T lymphocyte deficient horses with severe combined immunodeficiency (SCID). T lymphocytes appeared as early as the 75th day of fetal development and were normally distributed prior to birth of normal foals. Analysis of thymic T lymphocyte differentiation in SCID foals revealed the presence of both prothymocytes and mature thymocytes, but a virtual absence of cortical thymocytes. The data obtained support the hypothesis that two distinct pathways of T lymphocyte differentiation exist within the thymus. Although the gene defect in foals with SCID blocks the production of mature B and T lymphocytes, such foals do possess large granular lymphocytes which are cytotoxic following induction with interleukin 2. This suggests that lymphoid cells with natural killer cell activity are spared by the gene defect resulting in SCID in horses.     

Induction of B- and T-cell responses to cruzipain in the murine model of Trypanosoma cruzi infection

Trypanosoma cruzi, the causative agent of Chagas' disease, is an important cause of heart disease in Latin America. The parasite is transmitted mucosally, with both intra- and extracellular life stages in the human host. Cruzipain, the major cysteinyl proteinase of T. cruzi, has been shown to be antigenic in both humans and mice during infection with the parasite. We extend these observations, showing here that multiple murine immune subsets of potential importance for vaccine-induced protection can be induced by cruzipain. Cruzipain-specific serum IgG responses were induced during chronic infection with T. cruzi. In addition, T. cruzi mucosal infection stimulated the development of cruzipain-specific secretory IgA detectable in fecal extracts from infected mice. Cruzipain-specific type 1 cytokine responses characterized by the production of IFN-gamma but not IL-4 were also detectable during murine infection. Furthermore, immunization of mice with a DNA vaccine encoding cruzipain was shown to stimulate cytotoxic T lymphocyte (CTL) responses capable of recognizing and lysing T. cruzi-infected cells. The induction of serum antibody, mucosal IgA, Th1 cytokine and CTL responses by cruzipain in mice supports the use of this parasite protein for further efforts in T. cruzi vaccine development.