Maternal transmission of immunity to Eimeria maxima: enzyme-linked immunosorbent assay analysis of protective antibodies induced by infection.

Vaccination of broiler chickens against Eimeria infection is problematic because of the need to ensure that birds are protected from the time of hatching. We have therefore investigated the feasibility of protecting hatchling broilers via maternal transfer of protective antibodies from hens to their offspring. Oral infection of broiler breeder hens with 20,000 sporulated Eimeria maxima oocysts caused production of antibodies which were passed into the egg yolk and subsequently to hatchlings. The level of specific antibodies in the yolks to unsporulated oocysts, sporulated oocysts, merozoites, and gametocytes was assessed by enzyme-linked immunosorbent assays. The levels in yolks of antibodies to all developmental stages peaked 3 to 4 weeks after infection of the hens. Groups of 10 hatchlings were challenged at 3 days of age by oral infection with 100 sporulated E. maxima oocysts. In the first experiment, the mean 4-day (days 6 to 9 post-infection) total number of oocysts excreted in the feces of chicks from eggs collected 3 weeks after infection of the hens was (0.6 +/- 0.4) x 10(6) (mean +/- standard error) compared with (9.9 +/- 1.4) x 10(6) for the progeny of uninfected hens, which represents a greater than 90% reduction. However, oocyst excretion by chicks from eggs collected 7 or 8 weeks after infection of the hens was only 47 or 68% lower than control values, reflecting declining levels of protective antibodies. In a second experiment, in which the hens were somewhat older and pretreated by intramuscular injection of saline in the emulsifying agent, Arlacel A, the period for which protective antibodies were transferred to hatchlings was prolonged. Thus, oocyst excretion by challenged hatchlings from eggs collected for an 8-week period after infection of the hens was more than 90% lower than oocyst excretion by control chicks, and even hatchlings of eggs collected 19 weeks after infection of the hens showed a 60% reduction in oocyst output. In both experiments, the levels of immunoglobulin G (IgG) antibodies to all developmental stages in yolks or hatchling sera were very strongly correlated with maternally derived immunity to E. maxima. In contrast, parasite-specific IgM or IgA was not detectable, either in egg yolk or egg white. These results demonstrate the ability of IgG antibodies to protect against E. maxima in poultry, thus raising the possibility of using protective maternally derived IgG antibodies to identify potentially protective parasite antigens and indicating the feasibility of using maternal immunization as a means for parasite control.     

Maternal immunization with gametocyte antigens as a means of providing protective immunity against Eimeria maxima in chickens.

In the present study, we wished to demonstrate the ability of surface gametocyte antigens to induce protective immunity against Eimeria maxima infections in chickens. In order to accomplish this goal, we employed maternal immunization as a means of providing large amounts of specific antibodies to offspring chicks. Upon challenge with sporulated E. maxima oocysts, chicks from hens immunized with affinity-purified gametocyte antigens showed greatly reduced oocyst production compared with chicks from sham-immunized hens. These results suggest that maternal immunization with gametocyte antigens can be used as a means to provide transmission-blocking immunity against E. maxima infections.     

Induction of protective immunity against Eimeria tenella, Eimeria maxima, and Eimeria acervulina infections using dendritic cell-derived exosomes

This study describes a novel immunization strategy against avian coccidiosis using exosomes derived from Eimeria parasite antigen (Ag)-loaded dendritic cells (DCs). Chicken intestinal DCs were isolated and pulsed in vitro with a mixture of sporozoite-extracted Ags from Eimeria tenella, E. maxima, and E. acervulina, and the cell-derived exosomes were isolated. Chickens were nonimmunized or immunized intramuscularly with exosomes and subsequently noninfected or coinfected with E. tenella, E. maxima, and E. acervulina oocysts. Immune parameters compared among the nonimmunized/noninfected, nonimmunized/infected, and immunized/infected groups were the numbers of cells secreting T(h)1 cytokines, T(h)2 cytokines, interleukin-16 (IL-16), and Ag-reactive antibodies in vitro and in vivo readouts of protective immunity against Eimeria infection. Cecal tonsils, Peyer's patches, and spleens of immunized and infected chickens had increased numbers of cells secreting the IL-16 and the T(h)1 cytokines IL-2 and gamma interferon, greater Ag-stimulated proliferative responses, and higher numbers of Ag-reactive IgG- and IgA-producing cells following in vitro stimulation with the sporozoite Ags compared with the nonimmunized/noninfected and nonimmunized/infected controls. In contrast, the numbers of cells secreting the T(h)2 cytokines IL-4 and IL-10 were diminished in immunized and infected chickens compared with the nonimmunized/noninfected and the nonimmunized/infected controls. Chickens immunized with Ag-loaded exosomes and infected in vivo with Eimeria oocysts had increased body weight gains, reduced feed conversion ratios, diminished fecal oocyst shedding, lessened intestinal lesion scores, and reduced mortality compared with the nonimmunized/infected controls. These results suggest that successful field vaccination against avian coccidiosis using exosomes derived from DCs incubated with Ags isolated from Eimeria species may be possible.     

Construction of DNA vaccines and their induced protective immunity against experimental Eimeria tenella infection

In an attempt to construct a DNA vaccine against chicken coccidiosis, the TA4 gene of Eimeria tenella strain BJ was ligated to the mammalian expression vector pcDNA3.1/Zeo(+) to give pcDNA3.1-TA4 (pcDT). Then, Et1A (E. tenella refractile body gene) was ligated to it, upstream, aiming to be expressed in fusion with TA4, giving pcDNA3.1-Et1A-TA4 (pcDET). The constructed DNA vaccines were given to broilers intramuscularly 10–15 min after the breasts had been pre-treated with 25% sucrose solution. At 7 days after the second vaccination, chickens were challenged with 3×10⁴ sporulated oocysts of E. tenella BJ. The chickens were killed and the lesion scores of the ceca, the relative body-weight gains and the numbers of oocysts in the ceca of each group of chickens were calculated at day 8 post-inoculation. Results indicated that both pcDT and pcDET could induce protective immunity against coccidial challenge. Their use could obviously reduce oocyst output and alleviate chicken body-weight decrease due to coccidial infection. An anti-coccidial index of 160 was achieved with a treatment of 50 µg pcDET and 100 µg pcDT.     

Cell-mediated immunity in human herpesvirus infection: analysis by monoclonal antibodies

Peripheral blood lymphocytes (PBL) were obtained from heterosexual patients during the first or recurrent episodes of genital herpes simplex type 2 (HSV-2) infections. These cells were incubated with a panel of lymphocyte-specific monoclonal antibodies and examined by indirect immunofluorescence and flow cytometry. Lymphocyte proliferative responses to inactivated HSV-2 (UV-HSV-2) were also determined on repeat occasions using PBL obtained from patients with first episodes of infection. Flow cytometry analysis indicated that PBL obtained within 1 week after the onset of symptoms of first episode genital herpes exhibited significantly lower OKT 4 helper/OKT 8 suppressor T-cell ratios (1.26 +/- 0.12) than cells from either normal controls (2.44 +/- 0.3, P less than 0.01) or patients with recurrent genital HSV-2 infection (2.58 +/- 0.40, P less than 0.01). This lowered ratio was due to a decrease in OKT 4-positive helper T cells present early during initial infection (30.2 +/- 2.9, P less than 0.01 compared to recurrent disease patients or controls) and was not caused by a concomitant increase in OKT 8-positive T cells. No variation was observed during the course of infection in the proportion of total T cells as determined by EAET rosette formation or reactivity with the T-cell specific monoclonal antibody 9.6. PBL obtained from patients within 1 week after symptom onset exhibited a poor proliferative response to UV-HSV-2. Maximal proliferative responses occurred 6-9 weeks after disease onset and required the presence of OKT 4-positive T lymphocytes. The results of these experiments indicate that at different stages of disease, HSV infection is accompanied by fluctuations in the ratio of helper to suppressor T cells and alterations in antigen-specific lymphocyte proliferation. These findings suggest that variations in lymphocyte proliferative responses during the course of first episode genital herpes infection may reflect disease related variations in either the number or activity of circulating OKT 4-positive T lymphocytes.     

Acquisition of immunity to Eimeria maxima in newly hatched chickens reared on new or reused litter.

The acquisition of immunity by chickens infected 18 h post-hatch with 100 oocysts of Eimeria maxima and reared in floor-pens in contact with their droppings was investigated. In the first experiment, birds were placed on new litter and immunity was measured at 2, 3, 4, and 5 weeks by calculation of weight gain from days 0 to 7 following challenge with 100000 oocysts or by oocyst production in the faeces from days 5 to 8 following challenge with 500 oocysts. In the second experiment, birds were placed on new litter or reused litter from the first experiment (1 week after birds from the first experiment had been removed when 6 weeks of age), and were challenged at 1, 2, and 3 weeks of age. In the first experiment, immunity had developed in birds challenged at 3, 4, and 5 weeks, judged by weight gain and oocyst production, but immunity was not complete at 2 weeks. In the second experiment, immunity had developed in birds challenged at 1, 2, and 3 weeks measured by either criterion. In both experiments, birds produced small numbers of oocysts in their faeces following challenge. Judged by the weight gain following challenge, no significant difference in the acquisition of immunity was observed whether birds were reared on new or reused litter.     

Distinctive western blot antibody patterns induced by infection of mice with individual strains of the Mycobacterium avium complex.

Systemic infection of mice with organisms of the Mycobacterium avium complex (MAC) induced antibody responses, characteristic for each of the three tested individual strains. The influence of host genetic factors was reflected up to 3 months after infection by the finding of generally oligobanded and multibanded Western blot patterns in C57B1/6 and BALB/c mice, respectively. Nevertheless, more bands developed at 6 months in C57BL/6 mice. The response to three antigens of 18,000, 38,000 and 24,000 MW was analysed in greater detail. Antibodies to a protease-resistant 18,000 MW band produced only by BALB/c mice were either strain specific, following infection with M. avium, strain Maa-B2, or cross-reactive within MAC, following infection with M. avium strain Maa-A6 and M. paratuberculosis, strain Map-203. Another protease-resistant antigen of 38,000 MW was immunogenic only in Maa-B2 infected mice. This constituent was found to be related to the protease-sensitive antigen of corresponding molecular weight from M. tuberculosis. Two 24,000 MW proteins of M. paratuberculosis were separated by two-dimensional gel electrophoresis: antibodies to the anodic band were induced by Map-203 infection, whilst the cathodic band was revealed by heteroclitic antibodies from Maa-B2-infected mice. The latter antigen is apparently expressed during in vivo replication, but not during in vitro culture of Maa-B2 bacteria. We generally conclude, that the selective antibody patterns after live infection, could be attributed to differences in the release of native antigens within mycobacterial lesions. In view of a high degree of species specificity, some of the immunogenic constituents identified may also be useful for serodiagnostic application.     

Induction of protective immunity and neutralizing antibodies to pseudorabies virus by immunization of anti-idiotypic antibodies

Summary Xenogenic anti-idiotypic antibodies (anti-Id) were prepared in rabbits against three murine neutralizing monoclonal antibodies (MAbs) directed to pseudorabies virus glycoproteins. These anti-Id were highly specific to idiotopes on the corresponding MAb molecules. Because the binding of MAb to the corresponding anti-Id was inhibited by the addition of viral envelope protein, these anti-Id seemed to contain a subpopulation of antibodies against the antigen-combining site (paratope) or the region related to the paratope of the MAb molecules. One of the anti-Id to a MAb directed against glycoprotein gp50 induced neutralizing antibodies to PrV. Mice immunized with the anti-Id were protected from lethal infection of PrV.     

Mechanism of protective immunity induced by porin-lipopolysaccharide against murine salmonellosis.

Investigations were undertaken to characterize the protective immunity induced by porin-lipopolysaccharide (LPS) against Salmonella typhimurium infection in mice. Mice immunized with porin-LPS showed higher levels of antiporin immunoglobulin G than mice which received porin alone. Further, T cells from porin-LPS-immunized mice showed an augmented proliferative response to porin in vitro compared with the response of T cells from porin-injected animals. The passive transfer of anti-LPS antibodies conferred significant protection (17%), while antiporin serum failed to protect mice against lethal challenge, indicating the protective ability of anti-LPS antibodies. However, the transfer of serum obtained from porin-LPS-immunized mice resulted in better protection (30%) than did anti-LPS or antiporin antibodies alone. In contrast to LPS, monophosphoryl lipid A completely failed to induce protection against lethal infection. However, comparable to the effect of LPS, injection of porin with monophosphoryl lipid A enhanced antibody response and the protective ability of porin (81.25%). The transfer of T cells from porin-LPS-immunized mice provided higher levels of protection (47%) against lethal challenge than did T cells from porin-immunized mice (23%). The combination of T cells and serum from porin-immunized mice transferred 36% protection. However, a combination of T cells and serum from porin-LPS-immunized mice conferred the highest level of protection (92%), which was reflected by the number of survivors (100%) in the porin-LPS-immunized group. These results demonstrate that besides the protective effect of anti-LPS antibodies, the ability of LPS to augment humoral and cell-mediated immune responses to porin confers effective protection against Salmonella infection.     

Impairment of protective immunity to blood-stage malaria by concurrent nematode infection

Helminthiases, which are highly prevalent in areas where malaria is endemic, have been shown to modulate or suppress the immune response to unrelated antigens or pathogens. In this study, we established a murine model of coinfection with a gastrointestinal nematode parasite, Heligmosomoides polygyrus, and the blood-stage malaria parasite Plasmodium chabaudi AS in order to investigate the modulation of antimalarial immunity by concurrent nematode infection. Chronic infection with the nematode for 2, 3, or 5 weeks before P. chabaudi AS infection severely impaired the ability of C57BL/6 mice to control malaria, as demonstrated by severe mortality and significantly increased malaria peak parasitemia levels. Coinfected mice produced significantly lower levels of gamma interferon (IFN-gamma) during P. chabaudi AS infection than mice infected with malaria alone. Concurrent nematode infection also suppressed production of type 1-associated, malaria-specific immunoglobulin G2a. Mice either infected with the nematode alone or coinfected with the nematode and malaria had high transforming growth factor beta1 (TGF-beta1) levels, and concurrent nematode and malaria infections resulted in high levels of interleukin-10 in vivo. Splenic CD11c(+) dendritic cells (DC) from mice infected with malaria alone and coinfected mice showed similarly increased expression of CD40, CD80, and CD86, but DC from coinfected mice were unable to induce CD4(+) T-cell proliferation and optimal IFN-gamma production in response to the malaria antigen in vitro. Importantly, treatment of nematode-infected mice with an anthelmintic drug prior to malaria infection fully restored protective antimalarial immunity and reduced TGF-beta1 levels. These results demonstrate that concurrent nematode infection strongly modulates multiple aspects of immunity to blood-stage malaria and consequently impairs the development of protective antimalarial immunity.     

Monoclonal antibodies to baculovirus structural proteins: determination of specificities by Western blot analysis

Conventional mouse hybridoma technology was utilized to produce a panel of monoclonal antibodies which reacted with baculovirus proteins. Using an enzyme-linked immunosorbent assay (ELISA), the hybridomas which were raised against polyhedrin from Autographa californica nuclear polyhedrosis virus (AcNPV) and Choristoeura fumiferana nuclear polyhedrosis virus (CfNPV) were found to cross-react differentially with polyhedrins and granulins from several species of baculoviruses. Hybridoma antibodies which reacted against the nonoccluded form (NOV) of AcNPV in an ELISA test expressed different specificities for the occluded form of the virus (OV), a mutant strain of AcNPV, and CfNPV. Four hybridoma clones produced antibody which neutralized the infectivity of AcNPV NOV. One hybridoma antibody reacted strongly with the uninfected Spodoptera frugiperda host cell line. Using Western blot analysis, it was shown that hybridoma antibodies against polyhedrin reacted differentially with the complete polypeptide and protease-generated fragments of polyhedrin. The polypeptide specificity of 19 of 28 hybridoma antibodies which reacted with OV and NOV of AcNPV was assigned using Western blot analysis.     

Maternal to neonatal transmission of T-cell mediated immunity to Trichinella spiralis during lactation.

The potential of maternally derived cellular factors to mediate immunity to Trichinella spiralis in neonates during lactation was investigated in this study. Female FI rats, infected with T. spiralis, were able to transfer immunity to their suckling offspring, evidenced by a significant reduction in the intestinal parasite burdens of their neonates. When challenged between 2 and 3 weeks of age with 200 T. spiralis larvae, pups suckling on immune mothers harboured 28% and 26% (at 3 and 8 days post-challenge) of the worm numbers present in control neonates suckling on naive mothers. Cross-fostering experiments in which pups born of naive mothers but nursed by immune mothers showed significant immunity, demonstrated that this passage occurred through milk. The role of cell-mediated immunity in this immune transfer was analysed using T cells purified from MLN cells of syngeneic donor rats infected with T. spiralis. When 200 x 10(6) sensitized MLN T cells were adoptively transferred into lactating recipients, it led to the passive immunization of suckling neonates (26% and 13% of control values retained at 3 and 8 days post-challenge), while maternal injection of T cells primed to an irrelevant antigen (KLH) had no effect on neonatal immunity. Neonates fed per-orally with primed T lymphocytes early in lactation and prior to challenge were also rendered immune (34% and 44% of control values retained at 3 and 8 days post-challenge). A single dose of T. spiralis-primed T cells given to neonates in early lactation was sufficient to elicit a significant immune response in them at 2 weeks of age. These results support the hypothesis that cellular immunity mediated by antigen-specific T cells in milk can provide functional immune protection to the neonate against an intestinal pathogen.     

Serological diagnosis of HIV infection: incidence, outcome and significance of partial reactions found in western blot analysis.

Between May 1986 and April 1987 routine screening for anti-HIV antibody was performed by enzyme immunoassay (EIA) on 3344 serum samples. The 1160 samples found positive or borderline were further analysed in Western blot (Wb). We analysed the frequency of different patterns of 'intermediate' Wb reactions (1-3 'specific' bands) and tried to determine their significance by searching for possible modifications of the pattern of reaction a few months later. Of 1160 Wb, 461 were clearly positive, 489 negative and 210 'intermediate'. The latter consisted of: 92 sera with anti-p24 (associated or not), 23 with anti-gp 120 and 160, 16 with anti-p55, 12 with anti-p41, 10 with anti-p65, eight with anti-p17 and four with anti-p31. A non-specific pattern was observed in the remaining 45. Of these sera, 46% were obtained from high risk subjects, 38% from persons without risk and in 16% no reliable information was available. In 30 subjects (24 with p24 and 6 with p41), a second sample was obtained about three months later. The reaction persisted in nine, was replaced by another in five, and disappeared in 15. One subject with anti-p41 in the first sample became clearly positive. In one of the 15 samples with disappearance of the reaction, the antigen p24 was present as the only sign of HIV infection. Later samples of this subject showed clear seroconversion. In many subjects with and without risk of exposure to HIV, the Wb gives an intermediate pattern of reactions (1-3 specific bands), that does not permit definitive conclusion on one single sample. Later controls are therefore necessary. Most of these reactions do not correspond to HIV infection.     

Mechanisms of immunity in typhus infection: analysis of immunity to Rickettsia mooseri infection of guinea pigs.

To study the mechanisms of immunity to Rickettsia mooseri (R. typhi) infection, sera and splenic cells collected from nonimmune and immune guinea pigs were inoculated separately into syngeneic nonimmune recipients which were subsequently challenged intradermally. Protection was measured by comparing the course of the challenge infections of recipients with infections initiated with the same rickettsial inocula in nonimmune animals. Recipients of splenic cells collected 21 days after donor infection were protected from lesion development at sites of intradermal challenge and showed fewer rickettsiae in their kidneys. Cells obtained from nonimmune donors did not protect against either skin lesion development at sites of challenge or kidney infection. Antibody-containing sera collected 21 days after donor infection, but not normal sera, reduced levels of kidney infection, but immune sera did not protect against the development of lesions at sites of intradermal challenge. It was concluded that both immune sera and immune splenic cells possess capacities to effect a partial control of the systemic phase of R. mooseri infection in guinea pigs, but that immune splenic cells possess a capacity not shared by immune sera, i.e., the capacity to protect from infection at local sites of intradermal inoculation.     

Histopathological study of protective immunity against murine salmonellosis induced by killed vaccine.

Swiss-Webster mice were vaccinated with heat-killed salmonellae and then were infected with virulent Salmonella typhimurium. Only 1 of the 18 vaccinated mice died from a challenge of 10(4) X the 50% lethal dose, and about 70% of them survived a challenge of 10(5) X the 50% lethal dose. Histopathological examinations of the lesions developed in these vaccinated mice showed that they followed the characteristic features of a primary lesion in murine salmonellosis. There was an early necrosis with infiltration of polymorphonuclear leukocytes and abscess formation within the first 6 to 7 days after infection. However, these abscesses remained small and discrete. By days 7 to 10, the lesions began to transform into granulomas, first with the appearance of peripheral mononuclear cells and then by the replacement of polymorphs. By the third week of the infection, minute and discrete granulomas were seen scattered in the spleen, liver, and lymph nodes. Beyond this stage, healing and tissue regeneration followed. Thus, the characteristics of infectious lesions developed in mice vaccinated with heat-killed salmonellae are distinctly different from those developed in mice protected by the avirulent vaccine.     

Investigation of immunofluorescence cross-reactions against Trichinella spiralis by western blot (immunoblot) analysis.

Immunofluorescence cross-reactions in Trichinella spiralis serodiagnosis are sometimes difficult to identify. We compared the results of an indirect immunofluorescence assay and the profiles obtained by Western blot (immunoblot) analysis for three groups of patients: 10 T. spiralis-infected patients, 10 patients with autoimmune diseases, and 7 patients with parasitic diseases other than trichinellosis. The degree of immunofluorescence cross-reaction was variable. Western blotting allowed us to differentiate Trichinella infection from other parasitic diseases. In 3 of 10 serum samples from patients with autoimmune diseases, bands which had the same sizes as Trichinella bands were observed, and they could correspond to shared epitopes such as heat shock proteins.