Extended safety and efficacy studies of the attenuated Brucella vaccine candidates 16 M(Delta)vjbR and S19(Delta)vjbR in the immunocompromised IRF-1-/- mouse model.

The global distribution of brucellosis and high incidence in certain areas of the world warrant the development of a safer and efficacious vaccine. For the past 10 years, we have focused our attention on the development of a safer, but still highly protective, live attenuated vaccine for human and animal use. We have demonstrated the safety and protective efficacy of the vaccine candidates 16 MΔvjbR and S19ΔvjbR against homologous and heterologous challenge in multiple immunocompetent animal models, including mice and deer. In the present study, we conducted a series of experiments to determine the safety of the vaccine candidates in interferon regulatory factor-1-knockout (IRF-1(-/-)) mice. IRF-1(-/-) mice infected with either wild-type Brucella melitensis 16 M or the vaccine strain Brucella abortus S19 succumb to the disease within the first 3 weeks of infection, which is characterized by a marked granulomatous and neutrophilic inflammatory response that principally targets the spleen and liver. In contrast, IRF-1(-/-) mice inoculated with either the 16 MΔvjbR or S19ΔvjbR vaccine do not show any clinical or major pathological changes associated with vaccination. Additionally, when 16 MΔvjbR- or S19ΔvjbR-vaccinated mice are challenged with wild-type Brucella melitensis 16M, the degree of colonization in multiple organs, along with associated pathological changes, is significantly reduced. These findings not only demonstrate the safety and protective efficacy of the vjbR mutant in an immunocompromised mouse model but also suggest the participation of lesser-known mechanisms in protective immunity against brucellosis.

Protective efficacy and safety of Brucella melitensis 16MΔmucR against intraperitoneal and aerosol challenge in BALB/c mice.

Brucellosis is a zoonosis of nearly worldwide distribution. Vaccination against this pathogen is an important control strategy to prevent the disease. Currently licensed vaccine strains used in animals are unacceptable for human use due to undesirable side effects and modest protection. Substantial progress has been made during the past 10 years toward the development of improved vaccines for brucellosis. In part, this has been achieved by the identification and characterization of live attenuated mutants that are safer in the host but still can stimulate an adequate immune response. In the present study, the identification and characterization of the mucR mutant (BMEI 1364) as a vaccine candidate for brucellosis was conducted. BALB/c mice were vaccinated intraperitoneally at a dose of 10(5) CFU with the mutant to evaluate safety and protective efficacy against intraperitoneal and aerosol challenge. All animals vaccinated with the vaccine candidate demonstrated a statistically significant degree of protection against both intraperitoneal and aerosol challenge. Safety was revealed by the absence of Brucella associated pathological changes, including splenomegaly, hepatomegaly, or granulomatous disease. These results suggest that the 16MΔmucR vaccine is safe, elicits a strong protective immunity, and should be considered as a promising vaccine candidate for human use.

Amplified fragment length polymorphism reveals specific epigenetic distinctions between Mycobacterium avium subspecies paratuberculosis isolates of various isolation types.

Amplified fragment length polymorphism (AFLP) was employed as a genetic analysis tool for the study of the genetic relatedness of Mycobacterium avium subsp. paratuberculosis isolates harvested from bovine fecal samples and from bovine or human tissues. This analysis revealed genetic differences between these two isolate types that were confirmed through cluster analysis. Dendrogram analysis separated these two isolate types based on the isolation scheme (tissue-associated versus fecal M. avium subsp. paratuberculosis isolates). Further sequence analysis of unique genetic regions from each isolation type revealed no genetic sequence differences. However, Clustal DNA alignments identified AFLP restriction enzyme sites that were undigested in the tissue-associated isolates. AFLP analysis also disclosed that the same AFLP restriction sites were digested in all of the fecal isolates. Sequence analysis further revealed a consensus sequence upstream of the undigested restriction sites for possible methyltransferase recognition in the tissue-associated M. avium subsp. paratuberculosis isolates.

Early phase morphological lesions and transcriptional responses of bovine ileum infected with Mycobacterium avium subsp. paratuberculosis.

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of chronic enteritis in ruminants (Johne's disease) and a possible etiopathologic agent in human Crohn's disease. The host-pathogen interaction in this chronic disease has largely depended on the randomly collected static lesions studied in subclinically or clinically infected animals. We have established and utilized the neonatal calf ligated ileal loop model to study the early temporal host changes during MAP infection. After inoculation of ligated ileal loop with MAP, samples were analyzed for bacterial invasion, histologic and ultrastructural morphologic changes, and gene expression at several times (0.5-12 hours) postinfection. Our results indicate that MAP invades the intestinal mucosa as early as 0.5 hour postinoculation. Distribution and migration of neutrophils, monocytes/macrophages, and goblet cells were confirmed by histopathology, scanning and transmission electron microscopy. Coincident with the morphologic analysis, we measured by real-time polymerase chain reaction gene expression of various cytokines/chemokines that are involved in the recruitment of mononuclear and polymorphonuclear leukocytes to the site of infection. We also detected expression of several other genes, including intestinal-trefoil factor, profilin, lactoferrin, and enteric ss-defensin, which may play significant roles in the early MAP infection. Thus, the calf ligated intestinal loop model may be used as a human disease model to understand the role of MAP in the pathogenesis of Crohn's disease.

The Brucella abortus S19 DeltavjbR live vaccine candidate is safer than S19 and confers protection against wild-type challenge in BALB/c mice when delivered in a sustained-release vehicle.

Brucellosis is an important zoonotic disease of nearly worldwide distribution. Despite the availability of live vaccine strains for bovine (S19, RB51) and small ruminants (Rev-1), these vaccines have several drawbacks, including residual virulence for animals and humans. Safe and efficacious immunization systems are therefore needed to overcome these disadvantages. A vjbR knockout was generated in the S19 vaccine and investigated for its potential use as an improved vaccine candidate. Vaccination with a sustained-release vehicle to enhance vaccination efficacy was evaluated utilizing the live S19 DeltavjbR::Kan in encapsulated alginate microspheres containing a nonimmunogenic eggshell precursor protein of the parasite Fasciola hepatica (vitelline protein B). BALB/c mice were immunized intraperitoneally with either encapsulated or nonencapsulated S19 DeltavjbR::Kan at a dose of 1 x 10(5) CFU per animal to evaluate immunogenicity, safety, and protective efficacy. Humoral responses postvaccination indicate that the vaccine candidate was able to elicit an anti-Brucella-specific immunoglobulin G response even when the vaccine was administered in an encapsulated format. The safety was revealed by the absence of splenomegaly in mice that were inoculated with the mutant. Finally, a single dose with the encapsulated mutant conferred higher levels of protection compared to the nonencapsulated vaccine. These results suggest that S19 DeltavjbR::Kan is safer than S19, induces protection in mice, and should be considered as a vaccine candidate when administered in a sustained-release manner.

Effect of mean diameter and polydispersity of PLG microspheres on drug release: experiment and theory.

The need to tailor release rate profiles from polymeric microspheres is a significant problem. Microsphere size, which has a significant effect on drug release rate, can potentially be varied to design a controlled drug delivery system with desired release profile. In this work the effects of microspheres mean diameter, polydispersity, and polymer degradation on drug release rate from poly(lactide-co-glycolide) (PLG) microspheres are described. Piroxicam containing PLG microspheres were fabricated at 20% loading, and at three different impeller speeds. A portion of the microspheres was then sieved giving five different size distributions. In vitro release kinetics were determined for each preparation. Based on these experimental results, a suitable mathematical theory has been developed that incorporates the effect of microsphere size distribution and polymer degradation on drug release. We show from in vitro release experiments that microsphere size has a significant effect on drug release rate. The initial release rate decreased with an increase in microsphere size. In addition, the release profile changed from first order to concave-upward (sigmoidal) as the microsphere size was increased. The mathematical model gave a good fit to the experimental release data. For highly polydisperse populations (polydispersity parameter b<3), incorporating the microsphere size distribution into the mathematical model gave a better fit to the experimental results than using the representative mean diameter. The validated mathematical model can be used to predict small-molecule drug release from PLG microsphere populations.

Amplified fragment length polymorphism reveals genomic variability among Mycobacterium avium subsp. paratuberculosis isolates.

Ninety-six primer sets were used for amplified fragment length polymorphism (AFLP) to characterize the genomes of 20 Mycobacterium avium subsp. paratuberculosis field isolates, 1 American Type Culture Collection (ATCC) M. avium subsp. paratuberculosis isolate (ATCC 19698), and 2 M. avium subsp. avium isolates (ATCC 35716 and Mac 104). AFLP analysis revealed a high degree of genomic polymorphism among M. avium subsp. paratuberculosis isolates that may be used to establish diagnostic patterns useful for the epidemiological tracking of M. avium subsp. paratuberculosis isolates. Four M. avium subsp. paratuberculosis-polymorphic regions revealed by AFLP were cloned and sequenced. Primers were generated internal to these regions for use in PCR analysis and applied to the M. avium subsp. paratuberculosis field isolates. An appropriate PCR product was obtained in 79 of 80 reactions, while the M. avium subsp. avium isolates failed to act as templates for PCR amplification in seven of eight reactions. This work revealed the presence of extensive polymorphisms in the genomes of M. avium subsp. paratuberculosis and M. avium subsp. avium, many of which are based on deletions. Of the M. avium subsp. paratuberculosis-specific sequences studied, one revealed a 5,145-bp region with no homologue in the M. avium subsp. avium genome. Within this region are genes responsible for integrase-recombinase function. Three additional M. avium subsp. paratuberculosis-polymorphic regions were cloned, revealing a number of housekeeping genes; all were evaluated for their diagnostic and epidemiological value.

On the diameter and size distributions of Bovine Serum Albumin (BSA)-based microspheres.

A fluid mechanics-based correlation for the average size of Bovine Serum Albumin (BSA) microspheres, prepared using a water-in-oil emulsion technique, is presented. The correlation is formulated based on the theory of turbulent dispersion and a non-dimensional Weber number. Measured average diameters of the BSA microspheres prepared in olive oil at different stirring speeds are used to construct the correlation formula. The correlation gives good agreement with experimentally measured average diameters for a wide range of Weber numbers. This correlation is particularly useful to the pharmaceutical industry for predicting the size of encapsulated microspheres used in drug delivery prior to microsphere preparation. The effect of additives on microsphere size was also explored. The average diameter of the BSA microspheres was doubled by the addition of a bioadherent vitelline protein B solution. In addition, a Rosin-Rammler statistical distribution function is shown to accurately represent the microsphere size distribution obtained at different stirring speeds.

A novel 20-kilodalton protein conserved in Babesia bovis and B. bigemina stimulates memory CD4(+) T lymphocyte responses in B. bovis-immune cattle.

Acquired immunity against the hemoprotozoan parasite Babesia bovis is believed to depend on activation of antigen-specific CD4(+) T lymphocytes and IFN-gamma production. A strategy was employed to identify potentially protective antigens from B. bovis based on memory CD4(+) T lymphocyte recognition of fractionated merozoite proteins. Fractions of merozoites separated by continuous flow electrophoresis (CFE) that contained proteins of approximately 20 kDa were shown previously to stimulate memory CD4(+) lymphocyte responses in B. bovis-immune cattle with different MHC class II haplotypes. Expression library screening with rabbit antiserum raised against an immunostimulatory 20-kDa CFE fraction identified a 20-kDa protein (Bbo20) that contains a B lymphocyte epitope conserved in geographically distant B. bovis strains. An homologous 20-kDa protein that has 86.4% identity with Bbo20 and contains the conserved B cell epitope was identified in B. bigemina (Bbg20). Southern blot analysis indicated that both Babesia proteins are encoded by a single gene. Antibody against recombinant Bbo20 protein identified the antigen in CFE fractions shown previously to stimulate memory T lymphocyte responses in immune cattle. To verify Bbo20 as an immunostimulatory T lymphocyte antigen, CD4(+) T cell lines were propagated from B. bovis-immune cattle with merozoite antigen and shown to proliferate significantly against recombinant Bbo20 protein. Furthermore, Bbo20-specific CD4(+) T cell clones proliferated in response to several B. bovis strains and produced IFN-gamma. BLAST analysis revealed significant similarity of the Bbo20 and Bbg20 amino acid sequences with the hsp20/alpha-crystallin family.

A unique Babesia bovis spherical body protein is conserved among geographic isolates and localizes to the infected erythrocyte membrane.

Using monoclonal antibody (mAb) 70/52.9, generated from a Babesia bovis fraction enriched for spherical body organelles, we have identified a 135-kDa protein containing an epitope conserved in B. bovis strains from Texas, Mexico, and Australia. The protein was localized to the spherical bodies of the babesial apical complex and was designated spherical body protein 3 (SBP3), according to the established nomenclature. Immunofluorescence studies showed binding of the 70/52.9 mAb to the infected-erythrocyte membrane region but not to their uninfected counterparts, demonstrating a host-cell association shared with the previously isolated B. bovis spherical body proteins, SBP1 and SBP2. Using mAb 70/52.9, the full-length cDNA encoding SBP3 was isolated from an expression library, sequenced, and oligonucleotide primers synthesized to amplify the genomic copy by polymerase chain reaction. The genomic copy contained no introns and was identical to the cDNA sequence with each containing a single, large open reading frame encoding a protein of 1089 residues. Analysis of the SBP3 amino acid sequence revealed no significant amino acid identity to SBP1 and SBP2 and a lack of repeated epitopes, a notable feature of the other two spherical body proteins. Labeled probes derived from the coding region of SBP3 hybridized to single fragments on Southern blots containing B. bovis genomic DNA indicating a single copy gene. With the identification of this third spherical body protein, which associates with the cytoplasmic face of the infected-erythrocyte membrane, a complement of distinct B. bovis proteins have been identified that are likely to contribute to intracellular survival, growth, and development for this parasite. The encoded protein should be valuable for functional investigations and evaluation of potential targets for host immunity.

Bovine T cell responses to recombinant thioredoxin of Fasciola hepatica.

Fasciolosis is an economically significant disease of ruminants, caused by infection with the digenetic trematodes, Fasciola hepatica and F. gigantica. Some vaccination trials using irradiated metacercariae or isolated proteins have been shown to afford significant protection. However, the mechanisms of specific immunity against this pathogen have not been elucidated. We have identified thioredoxin, a tegument antigen of F. hepatica, among several proteins that are common to both the juvenile and adult fluke within the mammalian host and have undertaken studies to characterize bovine T cell responses to recombinant thioredoxin protein (FH 2020). Peripheral blood mononuclear cells from immune cattle proliferated specifically to crude F. hepatica antigenic extract but not to FH 2020. However, after repeated stimulation of lymphocytes by alternating crude extract and FH 2020, FH 2020-specific proliferation by T cell lines was observed. T cell clones were subsequently generated and found to respond specifically but weakly to both crude antigen and FH 2020. Thioredoxin appears to be only weakly antigenic for bovine T cells and is, therefore, an unpromising candidate for inducing resistance to F. hepatica.

Babesia bovis: common protein fractions recognized by oligoclonal B. bovis-specific CD4+ T cell lines from genetically diverse cattle.

CD4+ helper T cells are believed to be important for inducing protective immunity against Babesia bovis through the production of cytokines, including IFN-gamma, that will provide help to B lymphocytes for IgG production and activate macrophages to become parasiticidal. To provide maximum protection in an outbred population, an effective vaccine against B. bovis should contain antigens that would elicit an IFN-gamma response and would be recognized by cattle with diverse genetic backgrounds. To identify potentially protective "universal" T helper (Th) cell antigens, fractions of homogenized B. bovis merozoites were tested for the ability to stimulate proliferation of oligoclonal CD4+, IFN-gamma-producing T cell lines derived from four immune animals previously shown to differ in major histocompatibility complex class II expression. Homogenized B. bovis merozoites were separated by denaturing continuous flow electrophoresis (CFE) on 15, 10, and 7.5% polyacrylamide gels into fractions containing proteins ranging from <14.5 to approximately 95 kDa. Eighteen of 280 CFE fractions elicited anamnestic proliferative responses in all T cell lines tested. Nine of these cross-stimulatory fractions contained proteins of <14.5 to 24.5 kDa, and the remaining ones contained proteins with estimated molecular weights of 30, 31.5, 44.5, 49, 49.5, 54, 62, 72, and 82 kDa. Immunoblot analysis showed that four cross-stimulatory fractions contained a predicted known B. bovis antigen of similar molecular size. Previous studies had demonstrated that fractionated merozoite proteins stimulatory for CD4+ Th cell clones had apparent molecular weights similar to those present in 7 of the 18 stimulatory fractions. In the present study, two Th cell clones responded to cross-stimulatory CFE fractions, underscoring the potential to use both oligoclonal and monoclonal Th cell lines to identify commonly recognized polypeptides as potential vaccine antigens.

Bovine type 1 and type 2 responses.

The Th1/Th2 paradigm has provided a useful framework for understanding the observed bias in immune responses that are often dominated by either cell-mediated or humoral responses, and for devising therapeutic strategies to stimulate T cell- or antibody-mediated immunity. However this paradigm is an oversimplification of a much more complex immunoregulatory network. Studies with bovine Th cell clones and immunoregulatory cytokines support this viewpoint. This paper highlights the progress that has been made in defining type 1 and type 2 responses in cattle. Evidence is presented for the presence of different subtypes of antigen-specific Th cell clones of cattle which constitute a spectrum of cell phenotypes, and for cytokine-mediated regulation of Th cell responses that differs from that observed in mice. The majority of over 60 parasite antigen-specific Th cell clones coexpress IL-4 and IFN-gamma, and polarized cytokine profiles were rarely observed. Furthermore, IL-2 and IL-10 expression was not restricted to IFN-gamma or IL-4-producing cells, respectively. This lack of coordinate regulation of "Th1" and "Th2" cytokines strengthens the emerging viewpoint that Th1 and Th2 responses, per se, do not typify the immune response to most pathogens. In addition, we provide evidence that major regulatory cytokines, IL-4, IL-10, and IL-12, do not selectively exert their negative (IL-4 and IL-10) or positive (IL-12) effects on Th1-like cells.

Immunodominant T-cell antigens and epitopes of Babesia bovis and Babesia bigemina.

Despite convincing evidence that T cells are critical for both cellular and humoral immunity against haemoprotozoan parasites, the difficulty of performing meaningful experiments in cattle that would define the role of T cells in immunity to Babesia spp. has impeded research in this area. However, experiments performed ex vivo with immune T cells can reflect in-vivo events, and provide valuable insight into the nature of immunogenic proteins and the responding lymphocytes. The progress made towards identification of the immunogenic proteins and epitopes that stimulate anamnestic CD4+ type-1 (interferon-gamma-producing) T-cell responses in cattle immune to challenge with Babesia bovis or B. bigemina is the subject of the present review.

Sequence and characterization of phocine interleukin 2.

To improve assessment of cellular immune responses in seals, northern elephant seal (Mirounga angustirostris) interleukin 2 (IL-2) has been characterized. The gene was cloned and sequenced from a 658 base pair (bp) cDNA generated from total RNA by reverse transcription-polymerase chain reaction (RT-PCR). The sequence encoded a 154 amino acid (aa) polypeptide that included a 20 aa putative signal peptide. Seal IL-2 was found to share considerable identity with published sequences. Nucleotide sequence analysis of phocine (seal) IL-2 with canine, feline, human, trichechine (manatee), bovine and murine sequences demonstrated 93, 92, 86, 82, 78 and 71% identity, respectively. Analysis of the derived amino acid sequences demonstrated 88, 89, 78, 71, 66 and 60% identity, respectively. Interleukin-2 sequence identities appear to reflect evolutionary proximity among the analyzed species, and importantly, those residues identified as critical to IL-2 biological activity and receptor binding are largely conserved. To examine the kinetics of IL-2 mRNA expression, northern elephant seal lymphocytes were stimulated with the mitogen concanavalin A (Con A), and RNA was collected at several time points thereafter. The RT-PCR demonstrated that seal IL-2 mRNA expression peaks in the first 8 hr following Con A stimulation. Lastly, genomic DNA from northern elephant seal, harbour seal (Phoca vitulina) and California sea lion (Zalophus californianus) was used as template to identify and clone genomic IL-2. Partial sequence of the genomic clones demonstrated nearly complete identity among the three species. Sequence identity indicates that probes constructed from the northern elephant seal IL-2 gene will be effective in assessing IL-2 in other pinniped species.

Activation of intestinal intraepithelial T lymphocytes in calves infected with Cryptosporidium parvum.

The objective of this study was to identify disease-related changes in lymphocyte populations within ileal mucosae of calves with cryptosporidiosis. Groups of five neonatal calves were orally infected at 3 days of age with 10(8) oocysts and maintained in enteric-pathogen-free conditions until clinical disease was established or until the animals had recovered from disease. Age-matched uninfected calves were used for comparison. Ileal mucosal lymphocytes were collected, quantitated, and phenotyped to determine whether changes in lymphocyte composition occurred in infected animals. We observed significantly larger numbers of intraepithelial CD8+ T lymphocytes in ileal mucosae from acutely infected calves compared with those from control animals. In addition, a proportion of intraepithelial CD4+ T cells from acutely infected calves coexpressed CD25, whereas there was an absence of coexpressed CD25 on CD4+ T cells from control calves. Ex vivo reverse transcriptase PCR of RNA from intraepithelial lymphocytes from control calves showed a cytokine expression pattern consisting of tumor necrosis factor alpha (TNF-alpha) and gamma interferon (IFN-gamma), while intraepithelial lymphocytes from calves with cryptosporidiosis expressed IFN-gamma but not TNF-alpha. Together, the results indicate that changes occur in the ileal intraepithelial lymphocyte population coincidently with Cryptosporidium parvum-induced enteric disease.

Babesia bovis rhoptry-associated protein 1 is immunodominant for T helper cells of immune cattle and contains T-cell epitopes conserved among geographically distant B. bovis strains.

The ability of rhoptry-associated protein 1 (RAP-1) of Babesia bovis and Babesia bigemina to confer partial protective immunity in cattle has stimulated interest in characterizing both B-cell and T-cell epitopes of these proteins. It was previously shown that B. bovis RAP-1 associates with the merozoite surface as well as rhoptries and expresses B-cell epitopes conserved among otherwise antigenically different B. bovis strains. An amino-terminal 307-amino-acid domain of the molecule that is highly conserved in the B. bigemina RAP-1 homolog did not contain cross-reactive B-cell epitopes. The studies reported here demonstrate that B. bovis RAP-1 is strongly immunogenic for T helper (Th) cells from B. bovis-immune cattle and that like B-cell epitopes, Th-cell epitopes are conserved in different B. bovis strains but not in B. bigemina RAP-1. Lymphocytes from cattle immune to challenge with the Mexico strain of B. bovis proliferated against recombinant B. bovis RAP-1 protein derived from the Mexico strain. T-cell lines established by stimulating lymphocytes with recombinant RAP-1 protein responded against B. bovis, but not B. bigemina, merozoites. T-cell lines established by repeated stimulation of lymphocytes with B. bovis membrane antigen proliferated strongly against RAP-1, demonstrating the immunodominant nature of this protein. RAP-1-specific CD4+ T cell clones recognized Mexico, Texas, Australia, and Israel strains of B. bovis but neither B. bigemina merozoites nor recombinant B. bigemina RAP- 1. Analysis of cytokine mRNA in RAP-1-specific Th cell clones revealed strong expression of gamma interferon but little or no expression of interleukin-2 (IL-2), IL-4, or IL-10. Gamma interferon production was confirmed by enzyme-linked imunosorbent assay. These results indicate the potential to use selected B. bovis RAP-1 peptides as immunogens to prime for strong, anamnestic, strain-cross-reactive type 1 immune responses upon exposure to B. bovis.

Interleukin-10 downregulates proliferation and expression of interleukin-2 receptor p55 chain and interferon-gamma, but not interleukin-2 or interleukin-4, by parasite-specific helper T cell clones obtained from cattle chronically infected with Babesia bovis or Fasciola hepatica.

Human recombinant interleukin-10 (IL-10) was previously shown to inhibit accessory cell (AC)-dependent proliferation of bovine parasite-specific T helper 1 (Th1), Th2, and Th0 cells in an IL-2-reversible manner (Brown, W.C., Woods, V.M., Chitko-McKown, C.G., Hash, S.M., and Rice-Ficht, A.C., 1994. Infect. Immun. 62, 4697-4708). The present study was therefore designed to determine whether the effect of IL-10 on T cell proliferation corresponded with downregulated expression of cytokines, or their receptors, important for T cell growth. The effects of IL-10 on cellular proliferation and expression of IL-2, IL-4, IL-2 receptor (IL-2R; p55), and IFN-gamma by Babesia bovis- or Fasciola hepatica-specific Th cell clones were simultaneously evaluated. As shown previously, IL-10 strongly inhibited proliferation of all types of Th cell clones, although this did not correspond with reduced expression of IL-2 or IL-4 mRNA or their products. In contrast, expression of IL-2R mRNA was consistently reduced in the IL-10-treated clones. These results indicate that IL-10 does not inhibit AC-dependent proliferation of bovine Th cells by downregulating T cell cytokines; rather, IL-10 may act by downregulating IL-2R p55 expression and subsequent signal transduction leading to decreased cellular proliferation. IFN-gamma production was also consistently downregulated in the presence of IL-10.

Identification of Babesia bovis merozoite antigens separated by continuous-flow electrophoresis that stimulate proliferation of helper T-cell clones derived from B. bovis-immune cattle.

To characterize Babesia bovis merozoite antigens that stimulate anamnestic T helper (Th)-cell responses from B. bovis-immune cattle, B. bovis-specific Th-cell lines and clones, previously assigned to different antigenic groups (W. C. Brown, S. Zhao, A. C. Rice-Ficht, K. S. Logan, and V. M. Woods, Infect. Immun. 60:4364-4372, 1992), were tested in proliferation assays against fractionated merozoite antigens. The antigenic groups were determined by the patterns of response of Th clones to different parasite isolates and soluble or membrane forms of merozoite antigen. Soluble antigen fractionated by anion-exchange chromatography or gel filtration by using fast-performance liquid chromatography resolved two or three antigenic peaks, respectively. To enable fractionation of membrane-associated proteins and to resolve more precisely the proteins present in homogenized merozoites, a novel technique of continuous-flow electrophoresis was employed. Merozoite membranes or whole merozoites were homogenized and solubilized in sodium dodecyl sulfate-sample buffer, electrophoresed under reducing conditions on 15% or 10% acrylamide gels, eluted, and collected as fractions. Individual fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and tested for the ability to stimulate Babesia-specific CD4+ T-cell lines and clones. CD4+ Th-cell lines from two cattle displayed differential patterns of reactivity and detected numerous peaks of antigenic activity, ranging from < 14 to 76 kDa. Th-cell clones previously categorized into different antigenic groups detected antigenic peaks unique for clones representative of a given group. Antigens of 29, 51 to 52, and 85 to 95 kDa (group I), 40 kDa (group III), 20 kDa (group IV), 58 to 60 kDa (group VI), and 38, 45, and 83 kDa (group VII) were identified in the stimulatory fractions. Immunization of rabbits with selected fractions produced a panel of antisera that reacted specifically on Western blots (immunoblots) with merozoite antigens of similar sizes, leading to the tentative identification of candidate antigens of B. bovis merozoites with molecular masses of 20, 40, 44, 51 to 52 or 95, and 58 to 60 kDa that stimulate proliferation of Th clones representative of five different antigenic groups. These antisera may be useful for isolating recombinant proteins that are immunogenic for Th cells of immune cattle and therefore potentially useful for vaccine development.