Humoral immune responses and safety of experimental formulations of inactivated Neospora vaccines.

Antibody titers to Neospora antigens ranged from 40 to 160 before vaccination, from 80 to 5,120 2 weeks after the first dose of vaccination, and 320 to 40,9602 weeks after the second (booster) vaccination. A peak antibody titer of 40,960 was also detected 28 days after the booster vaccination among animals vaccinated with Neospora vaccine formulated with Bay R1005 adjuvant. In heifers inoculated with experimental formulations of Neospora vaccines, transient development of injection site reactions resulted in 1 out of 15 animals. This injection site reaction was not detectable 14 days after the first observation and measurements were made. We have also demonstrated that vaccines derived from tissue-culture-grown Neospora tachyzoites are safe and would be expected to be efficacious.

A POLYGEN-adjuvanted killed Neospora caninum tachyzoite preparation failed to prevent foetal infection in pregnant cattle following i.v./i.m. experimental tachyzoite challenge.

Cattle immunised with a POLYGEN-adjuvanted killed Neospora caninum tachyzoite preparation were previously shown to produce interferon (IFN)-gamma at levels similar to those of tachyzoite-infected cattle. In view of the critical role of IFN-gamma in resistance of mice to N. caninum infection, these results prompted us to test the POLYGEN-adjuvanted preparation in pregnant cattle to determine whether it will be able to prevent foetal infection following an experimental tachyzoite challenge. Seven heifers were immunised at 35 and 63 days of gestation with the POLYGEN-adjuvanted preparation, while five heifers were inoculated with POLYGEN alone at the same days of gestation. Four weeks later, all heifers were challenged with a combined i.v./i.m. inoculation of tachyzoites. The same challenge was given to seven unimmunized heifers at the same stage of gestation. An additional unimmunized heifer was inoculated with uninfected monolayer cell culture material. All challenged heifers, immunized and unimmunized, had infected foetuses. Immunized heifers developed both parasite-specific humoral and cellular immune responses, characterised by increased IFAT titres, a predominant IgG1 response, elevated lymphoproliferative response and IFN-gamma production. Following tachyzoite challenge, they developed an anamnestic humoral response and produced similar amounts of IgG1 and IgG2 antibodies, but did not have an anamnestic cellular immune response. The lack of anamnestic cellular immune response and/or the large i.v/i.m tachyzoite inoculum may have contributed to the failure of the preparation.

Immunogenicity of a killed whole Neospora caninum tachyzoite preparation formulated with different adjuvants.

A killed whole Neospora caninum tachyzoite preparation was formulated with various adjuvants and tested for its immunogenicity in cattle. The adjuvants used were: Havlogen, a polymer of acrylic acid cross-linked with polyallylsucrose; Polygen, a non-particulate copolymer; a mixture of Havlogen and Bay R-1005, which is a preparation of free base synthetic glycolipids; and Montanide ISA 773, a water-in-oil emulsion made with a mixture of metabolisable and mineral oils. Immune responses in immunised cattle were compared with those of cattle experimentally infected with culture-derived N. caninum tachyzoites. The overall mean serum IFAT titres were significantly higher (P < 0.05) in experimentally infected cattle compared with all immunised cattle. Nonetheless, the maximum antibody titres of the immunised cattle, which were obtained following the third immunisation, were within the range of titres previously described for naturally infected cattle. The overall mean serum IFAT titres were significantly higher (P < 0.05) in cattle immunised with the killed tachyzoite preparation formulated with Polygen and with the mixture of Havlogen and Bay R-1005, compared with cattle immunised with the Havlogen- and Montanide-based preparations. Two of the four adjuvant preparations were able to induce cell-mediated immune responses similar to those of the experimentally infected cattle. The Havlogen-adjuvanted tachyzoite preparation elicited N. caninum-specific proliferation of peripheral blood mononuclear cells statistically similar (P = 0.095) to that of the infected animals. Peripheral blood mononuclear cells from animals immunised with the Polygen-adjuvanted tachyzoite preparation produced interferon-gamma concentrations of similar magnitude (P = 0.17) to those from the infected animals. Polygen was one of two adjuvants that elicited the highest antibody responses, and was the only adjuvant that induced interferon-gamma levels similar to those of the infected heifers.

A field trial of the effectiveness of a feline Toxoplasma gondii vaccine in reducing T. gondii exposure for swine.

A 3-yr field trial was conducted on 8 commercial swine farms in Illinois to determine the effectiveness of a feline Toxoplasma gondii vaccine in reducing the exposure of swine to T. gondii. A vaccine consisting of live bradyzoites of the mutant T-263 strain, capable of preventing oocyst shedding by cats, was used in this study. Each farm was visited 3 times in 1994, 3 times in 1995, and once in 1996. Cats were trapped and inoculated with the T-263 oral vaccine during 1994 and 1995. On each visit, the following samples were collected: blood from pigs, cats, and mice for detection of serum antibodies to T. gondii, feces from cats to detect oocysts, and heart and brain tissues from rodents to determine the presence of T. gondii tissue cysts. The modified agglutination test (MAT), with a positive titer set at the 1:25 dilution, was used to determine serum antibodies. At first capture, 72.6% (61/84) of juvenile cats and 32.6% (31/95) of adult cats had no detectable antibodies (seronegative), indicating no prior exposure to T. gondii when they received their first vaccine. Of these first-time seronegative cats, 58.1% (18/31) of adult and 45.9% (28/61) of juvenile cats were recaptured and received a second dose of vaccine. Changes in the prevalence of T. gondii infection were evaluated from the prevaccination (1992, 1993) to the postvaccination (1996) period. Eleven cats (5%) were detected shedding oocysts between 1994 and 1996, of which 10 (90.1%) shed during 1994. The last detection of oocyst shedding by cats was during the first farm visit in 1995. There was a significant decrease in T. gondii seroprevalence for finishing pigs (P < 0.05, Wilcoxon sign rank test). There was a positive correlation (Spearman's p = 1.0, P < 0.0001) between the change in prevalence in juvenile cats and the change in prevalence in finishing pigs. The seropositivity rate (MAT > or = 1:25) in mice among all farms decreased from 4% in 1992-1993 to 0% in 1996. The mean prevalence of T. gondii tissue cyst isolation for mice on all farms decreased from 1.1% in 1994, to 0.8% in 1995, and to 0.5% in 1996. The results of this study suggest that the reduced exposure of pigs to T. gondii was due to the administration of the T. gondii vaccine to cats.

Immunization of cats with tissue cysts, bradyzoites, and tachyzoites of the T-263 strain of Toxoplasma gondii.

Previous studies have demonstrated that oral administration to cats of tissue cysts of the oocyst-negative mutant strain of Toxoplasma gondii, T-263, induces immunity to oocyst shedding following challenge. Experiments were designed to compare the levels of protection induced by T. gondii T-263 when tissue cysts, bradyzoites released from tissue cysts, and tachyzoites were administered to cats. In 1 experiment, groups of cats received 2 oral doses of intact tissue cysts or released bradyzoites of T. gondii T-263 and were challenged 47 days later with the oocyst-producing strain of T. gondii T-265. All cats seroconverted following immunization and none of them shed oocysts following challenge. In a second experiment, groups of cats received tachyzoites of T. gondii T-263 as a single oral dose and either 1 or 2 intraduodenal doses; they were challenged 60 days after the last vaccination. All cats seroconverted following immunization. Following challenge, all cats shed oocysts except for 2 of 7 cats that received 2 intraduodenal doses of tachyzoites. Thus, orally administered bradyzoites of T. gondii T-263, either contained in intact tissue cysts or liberated from cysts, induced immunity to oocyst shedding. In contrast, tachyzoites did not completely protect against oocyst shedding, even when delivered directly to the duodenum and despite the development of high antibody titers.

Augmentation of suppressed antibody responses in mice during experimental Chagas' disease by T helper cells activated in a time-dependent mode of immunization.

Mice infected with the protozoan parasite Trypanosoma cruzi, the causative agent of human Chagas' disease, develop immunosuppressed responses to heterologous antigens. Experiments were performed using infected mice in the acute stage of infection to assess immunoregulatory activities during induction of direct plaque-forming cells (DPFC) to sheep erythrocytes (SRBC), hapten-conjugated SRBC (TNP-SRBC), and horse erythrocytes (TNP-HRBC). Studies in vivo demonstrated that anti-SRBC responses were best enhanced when T. cruzi-infected mice were injected with primed T cells derived from normal or infected mice immunized four days previously. The presence of enhancing capacities for DPFC responses by T cells from T. cruzi-infected mice were also supported by experiments examining the hapten-carrier effect. Preimmunization of infected mice with SRBC or HRBC four days before injection of hapten-homologous (TNP-SRBC or TNP-HRBC) carrier resulted in markedly augmented anti-hapten antibody responses. These results show that functional help provided by T cells activated during priming and exposed to a challenge dose of antigen (SRBC) in a time-dependent mode can overcome the effect of immunosuppression in T. cruzi-infected mice.

Detection of antigens of larval Taenia solium in the cerebrospinal fluid of patients with the use of HPLC and ELISA.

The cerebrospinal fluid (CSF) obtained from patients suspected of having neurocysticerosis (79 samples), as well as from control patients (without neurological symptoms), was separated using a high performance liquid chromatography gel filtration column. During the chromatographic separation, the eluted fractions were collected separately according to distinctive peaks. The elution characteristics of CSF components were identified by aligning more than 100 chromatograms and 6 distinctive peaks, eluting in consistent positions. Samples of each peak were tested in an enzyme-linked immunosorbent assay (ELISA) for the presence of larval antigens. Forty-four of the suspected 79 cases were found to have larval antigens in their CSF and these antigens were detected in peak no. 2, the mean of which is approximately 110,000 molecular weight. Also, in some cases, larval antigens were found in peak no. 1; however, we were able to detect them in only 23 CSF samples out of 44 CSF samples in which larval antigens were present in peak no. 2. Nine of these 23 CSF samples (derived from 79 patients in which neurocysticercosis was suspected) were later confirmed by histopathology. Values of ELISA readings of 5 CSF samples obtained from control patients (0.054 +/- 0.064) were considered negative. Thus, in 44 of 79 CSF samples from patients suspected of having neurocysticercosis, the ELISA values were highly positive (0.551 +/- 0.293). The remaining 35 CSF samples gave ELISA readings of 0.092 +/- 0.062, which were not significantly different from values obtained with CSF of control patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Time-dependent mode of immunization augments suppressed antibody responses in spleen cell cultures from mice experimentally infected with Trypanosoma cruzi.

Mice infected with Trypanosoma cruzi develop immunosuppressed responses to heterologous antigens. Experiments were performed using infected mice in the acute stage of infection to assess immunoregulatory activities during induction of direct plaque-forming cells (DPFC) to sheep erythrocytes (SRBC). After normal or infected mice were primed with SRBC, their spleen cells were restimulated 4 days later with SRBC in Mishell-Dutton cultures and found to mount hyperaugmented IgM anti-SRBC responses. It was also demonstrated that T-cells derived from normal mice primed in vivo 4 days previously with SRBC, and subsequently added to cultures of spleen cells from T. cruzi-infected mice, enhanced anti-SRBC DPFC responses in a dose-dependent fashion. These results show that functional help provided by T-cells activated during an in vivo priming and exposed to an in vitro challenge dose of antigen (SRBC) in a time-dependent mode can overcome the effect of immunosuppression in the spleen cell cultures from T. cruzi-infected mice.

Relationship of route of infection to susceptibility and immune response of inbred mice to Y strain Trypanosoma cruzi.

C3HeB/FeJ (C3H) mice infected ip with 10(6), 5 x 10(5), and 10(5) blood-form trypomastigotes (BFTs) of the Y strain of Trypanosoma cruzi were more resistant than C57B1/6 (B6) mice infected in the same manner. This pattern of susceptibility is opposite that reported for other stocks of this parasite. In a second experiment, C3H and B6 mice were infected ip or sc with 2 x 10(6), 10(6), 5 x 10(5), 10(5), or 10(3) Y strain BFTs. C3H mice infected ip with the 3 highest doses were again more resistant than the B6 mice, while mice infected ip with the 2 lowest doses were essentially equivalent in resistance. Thus, the difference in susceptibility was detectable, in terms of parasitemia levels and survival, primarily at the higher infection doses. For the groups infected sc, the pattern of susceptibility reversed. B6 mice infected with the 3 highest doses had lower parasitemia levels than the corresponding C3H mice, while C3H and B6 mice infected with 10(5) or 10(3) BFTs were similar in resistance. Blastogenic responses of lymphoid cells to phytohemagglutinin (PHA) and a soluble trypanosome extract (STE) were compared for C3H mice infected ip or sc to determine if the susceptibility to infection obtained with the 2 routes would be associated with differences in immune responses. Mesenteric lymph node cells (MLNCs) of mice infected ip were responsive to the STE early in infection, while superficial lymph node cells (SLNCs) of these mice were not. C3H mice infected sc had SLNCs which yielded strong responses to STE, while their MLNCs were relatively unresponsive. PHA stimulated responses by lymphoid cells from mice infected ip or sc were similar.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of interleukin-2 on parasitemia and myocarditis in experimental Chagas' disease.

Previous investigations showed that interleukin-2 (IL-2) administered in vivo into mice infected with Trypanosoma cruzi reduced levels of parasitemia and increased longevity. Present experiments examined the effect of administration of different doses of IL-2 at different times during infection in mice on parasitemia and histopathology of heart tissue. Two different doses of IL-2 (1,500 or 10,000 U) given at 3 different times during infection were equivalent in reducing parasitemia. All of the IL-2 treated groups of mice had significantly lower numbers of circulating trypomastigotes as compared with controls not receiving this lymphokine. This IL-2 treatment of T. cruzi-infected mice resulted also in lower numbers of pseudocysts in all 4 ventricular regions in the hearts. This was particularly evident in the more severely infected right ventricular wall; however, a similar decrease was not as apparent in the less severely infected left ventricular wall. The IL-2 treated, infected mice showed minimal or no effect in reducing inflammation of myocardial cells. However, the mildest inflammation of ventricular wall tended to occur in mice receiving IL-2 treatment either as a low dose (1,500 U) or a high dose (10,000 U) at 5, 7 and 9 days after infection as compared with mice treated later on. It was concluded that IL-2 treatment of infected mice produced a significant decrease in parasitemia and decreased infection of myocardial cells.

Use of parasite antigens and interleukin-2 to enhance suppressed immune responses during Trypanosoma cruzi infection in mice.

Mice infected with Trypanosoma cruzi exhibit an early and profound suppression of parasite-specific and nonspecific immune responses. Earlier studies have shown that this suppression is due, at least in part, to suppressor macrophages, deficiency in production of interleukin-2 (IL-2), and reduced T helper (Th)-cell activity. In the present study, the effect of exogenously supplied IL-2 on enhancement of parasite-specific Th-cell activity, anti-parasite immunoglobulin G (IgG) and IgM antibody levels, parasitemia, and longevity was examined in infected mice. The results showed that administration of IL-2 with and without antigenic stimulation with trinitrophenylated T. cruzi significantly enhanced parasite-specific IgM and IgG levels. Injection of IL-2 and trinitrophenylated T. cruzi together significantly enhanced parasite-specific Th-cell activity and was more effective in enhancement of parasite-specific antibody levels. In addition, it was found that IL-2 alone had a rapid and lasting effect in reducing parasitemia. These results suggest that deficiency in IL-2 may play a major role in host susceptibility to T. cruzi.

Repeated antigenic stimulation overcomes immunosuppression in experimental Chagas' disease.

Mice infected with Trypanosoma cruzi, and profoundly suppressed in their ability to respond to sheep erythrocytes (SRBC), were found to become highly responsive to these antigens when given two injections of SRBC at 4- or 6-day intervals. Two injections at intervals of 2, 8 or 10 days did not restore responsiveness. The ability to overcome immunosuppression via two challenges with antigen was found to be antigen-specific, in that if the first injection was with SRBC and the second with horse RBC there was no enhancement of plaque-forming cells to either antigen. Homologous challenges with SRBC or HRBC, however, did overcome immunosuppression. It is suggested that the ability to overcome immunosuppression by two injections of antigens at 4- or 6-day intervals is due to stimulation of a small number of T-helper cells in the first injection and an expansion of these cells in the second injection resulting in sufficient help to induce specific B-cell responses.

Interleukin 2 enhances specific and nonspecific immune responses in experimental Chagas' disease.

Mice infected with Trypanosoma cruzi develop an early and profound immunosuppression of responses to heterologous antigens. Recently it has been demonstrated that this immunosuppression is linked, in part, to deficiency in the production of interleukin 2 (IL-2), and that the addition of IL-2 to cultures of normally unresponsive spleen cells from infected mice will restore responsiveness to sheep erythrocytes (SRBC) and enhance parasite-specific immune responses. In the present study, the effect of administration of ultrapure or recombinant IL-2 on immune responses to SRBC and parasite-specific responses in vivo was examined. It was found that a single injection of 1,500 U of IL-2 provided at the same time as SRBC more than doubled the number of direct plaque-forming cells to SRBC and that multiple injections of 1,500 U of IL-2 were no more restorative than a single injection. Anti-SRBC responses of normal mice were unaffected by injection of IL-2. Single or multiple injections of recombinant human IL-2, with and without gelatin, into highly susceptible C3H(He) mice induced greater parasite-specific immunity as reflected by significantly reduced levels of parasitemia and increased longevity. Three injections of 1,500 U each of recombinant human IL-2 on days 10, 14, and 18 was found to be the most efficacious in reducing parasitemia and increasing longevity. Injection of IL-2 with gelatin did not enhance the effect of IL-2 alone.

Production and characterization of clones of the Brazil strain of Trypanosoma cruzi.

Six clones and 4 subclones were isolated from the Brazil strain of Trypanosoma cruzi and were passaged in C3H(He) mice. Parasitemia levels and survival times of mice infected with 8 of the isolates were equivalent to the Brazil strain in virulence. Two clones, designated WFTc-5.1 and WFTc-6.1 (WFTc = Wake Forest Trypanosoma cruzi) were of lower virulence in C3H mice than the other isolates and the Brazil strain. C57BL/6 mice infected with WFTc-5.1 had significantly lower parasitemias and higher survival rates than C57BL/6 mice infected with the Brazil strain or a clone designated WFTc-3.2. Levels of anti-T. cruzi IgM and IgG antibodies were the same in mice infected with higher virulence or lower virulence isolates. Based on these results the Brazil strain of T. cruzi is composed of distinct subpopulations which are heterogeneous with respect to virulence.

Further studies on the surface saccharides in Trichomonas vaginalis strains by fluorescein-conjugated lectins.

Fluorescence emitted by individual cells of several Trichomonas vaginalis strains, nearly all of which were cloned, incubated with fluorescein-conjugated lectins in the absence (experimental) or presence (control) of inhibitory sugars, or else in phosphate-buffered saline alone (autofluorescence) was measured with a Leitz MPV Compact microfluorometer. Irrespective of whether the organisms were postfixed in formalin or glutaraldehyde, the relative fluorescence emitted by the cells was closely comparable, provided that appropriate neutral density filters were employed. However, autofluorescence was much higher for glutaraldehyde-fixed trichomonads. Therefore, although better preserved and more amenable to subsequent manipulations, such organisms were found unsuitable for use in "qualitative" titration of the fluorescence emitted by various strains. Provided that the necessary precautions were taken, comparable fluorescence readings were obtained with trichomonads affixed to glass slides by heat (41 degrees C, on a section spreader) or by a cytologic centrifuge (Cytospin 2). Large numbers of concanavalin A (Con A)-binding sites were present on organisms of all strains, irrespective of their virulence for human patients and as estimated by the subcutaneous mouse assay; these sites were shown with the aid of D-mannose to be mannose or mannose-related residues. More binding sites for soybean agglutinin (SBA) were found on the virulent than on avirulent strains. On the basis of inhibition experiments, the sugar residues mainly responsible for these differences appeared to be D-lactose residues. Similar differences were observed with Ricinus communis agglutinin Type I (RCA I), for which D-galactose was employed as the competing sugar. However, with two cloned strains the situation with regard to RCA I binding was reversed - more of the lectin bound to a mild than to a virulent strain. The results obtained with Ricinus communis Type II agglutinin (RCA II) were often similar to those noted for RCA I; however, in most instances the inhibition with N-acetyl-D-galactosamine (GalNAc) was lower. Furthermore, the results noted with the GalNAc-specific agglutinins from Dolichus biflorus and Helix pomatia suggested that only very few GalNAc residues were available for binding on the surfaces of all T. vaginalis strains examined in the course of this study. Statistical analyses of fluorescence emitted by four clones of each, Balt 42 (virulent) and JH31A (avirulent) T. vaginalis strain upon incubation with Con A and SBA revealed homogeneity of these strains with regard to the number of the specific surface saccharide residues, D-mannose and D-lactose.(ABSTRACT TRUNCATED AT 400 WORDS)