A nano particle vector comprised of poly lactic-co-glycolic acid and monophosphoryl lipid A and recombinant Mycobacterium avium subsp paratuberculosis peptides stimulate a pro-immune profile in bovine macrophages.

AIMS: We evaluated the potential of a nanoparticle (NP) delivery system to improve methods of delivery of candidate peptide-based vaccines for Paratuberculosis in cattle. METHODS AND RESULTS: Peptides derived from Mycobacterium avium subsp. paratuberculosis (Map), and the pro-inflammatory monophosphoryl lipid A (MPLA) were incorporated in polymeric NPs based on poly (d,l-lactide-co-glycolide) (PLGA). The PLGA/MPLA NPs carriers were incubated with macrophages to examine their effects on survival and function. PLGA/MPLA NPs, with and without Map antigens, are efficiently phagocytized by macrophages with no evidence of toxicity. PLGA/MPLA NP formulations did not alter the level of expression of MHC I or II molecules. Expression of TNFα and IL12p40 was increased in Map-loaded NPs. T-cell proliferation studies using a model peptide from Anaplasma marginale demonstrated that a CD4 T-cell recall response could be elicited with macrophages pulsed with the peptide encapsulated in the PLGA/MPLA NP. CONCLUSIONS: These findings indicate PLGA/MPLA NPs can be used as a vehicle for delivery and testing of candidate peptide-based vaccines. SIGNIFICANCE AND IMPACT OF THE STUDY: These results will assist on more in depth studies on PLGA NP delivery systems that may lead to the development of a peptide-based vaccine for cattle.

Bovine NK cells acquire cytotoxic activity and produce IFN-gamma after stimulation by Mycobacterium bovis BCG- or Babesia bovis-exposed splenic dendritic cells.

Early interactions of innate immune cell populations, such as dendritic cells (DC) and natural killer (NK) cells, can affect the ability of the acquired immune response to control infection of intracellular microorganisms. In this study, we investigated the activation of bovine NK cells by CD13(+) splenic DC stimulated with either Mycobacterium bovis BCG or Babesia bovis merozoites. Splenic DC were used either immediately after selection (cytokine(-)) or after exposure to GM-CSF, IL-4 and Flt3L for 72 h (cytokine(+)). Phenotypic analyses showed up-regulation of MHCII, CD80 and CD86 on cytokine(+) DC when compared to cytokine(-) DC. Purified NK cells (CD335(+)CD3(-)CD2(+/-)CD8alpha(+/-)) were co-cultured with microbial-exposed cytokine(-) DC or cytokine(+) DC in either transwell or cell-to-cell format and NK cell IFN-gamma production and cytotoxicity were assessed. NK cell IFN-gamma production was dependent on cell-to-cell contact. Microbial-stimulated cytokine(+) DC induced significantly more IFN-gamma production from NK cells than cytokine(-) cells. In contrast, cytotoxicity and perforin up-regulation were more pronounced in NK cells cultured with cytokine(-) DC than cytokine(+) DC. Therefore, activation of bovine NK cells by microbial-stimulated CD13(+) splenic DC is influenced by the maturation state of the DC suggesting different roles for the splenic DC during disease-induced maturation.

Differential response of splenic monocytes and DC from cattle to microbial stimulation with Mycobacterium bovis BCG and Babesia bovis merozoites.

Both bovine peripheral blood monocyte-derived dendritic cells (DC) and myeloid DC from afferent lymph have been described, but resident DC from other bovine tissues have not been fully characterized. The spleen as a secondary lymphoid organ is central to the innate and acquired immune response to various diseases particularly hemoprotozoan infections like babesiosis. Therefore, we developed methods to demonstrate the presence of myeloid DC from the spleen of cattle and have partially characterized a DC population as well as another myeloid cell population with monocyte characteristics. The phenotypic profile of each population was CD13+CD172a+/-CD14-CD11a-CD11b+/-CD11c+ and CD172a+CD13+/-CD14+CD11a-CD11b+/-CD11c+, respectively. The CD13+ population was found exclusively in the spleen whereas the CD172a+ population was present at the same percentage in the spleen and peripheral blood. CD13+ cells developed a typical veiled appearance when in culture for 96 h. The two cell populations differed in their ability to produce nitric oxide and had a different pattern of cytokine mRNA when stimulated with Mycobacterium bovis BCG or Babesia bovis merozoites. The data demonstrate the presence of a myeloid splenic DC with attributes consistent with an immature status.

Capacitance of bilayers in the presence of lipophilic ions.

The capacitance of glycerolmonooleate and egg phosphatidylcholine bilayer membranes in the presence of NaCl solutions containing tetraphenylborate, tetraphenylarsonium or dipicrylamine ions has been measured using alternating current techniques over a wide range of frequencies (1-200 kHz). The concentrations of ions corresponded to the lower limits of conductance saturation. Similar determinations were also made with solutions containing no lipophilic ions. The experimental method used in this work requires correction of admittance measurements for the solution resistance in series with the membrane, as well as careful area determinations. In all cases membrane capacitance levels off at sufficiently high frequencies to values which are independent of frequency. The high-frequency capacitance, which is regarded as the 'geometrical capacitance' due to dielectric polarization, is practically unaffected by the presence of lipophilic ions. The results support the assumption made in other studies, such as in charge pulse investigations, that the adsorption of lipophilic ions at concentrations up to the saturation range does not have an important effect on the dielectric properties of bilayers.

Development of detection methods for ruminant interleukin (IL)-4.

Recombinant bovine IL-4 (rbo IL-4) was transiently expressed in COS-7 cells. Mice were immunised with a plasmid encoding rbo IL-4 and boosted with rbo IL-4. A number of monoclonal antibodies (mAb) were generated that reacted with rbo IL-4 in an ELISA and these cloned hybridomas were termed CC311, CC312, CC313 and CC314. A pair of mAb (CC313 and CC314) was identified that together could be used to detect both recombinant and native bovine IL-4 by ELISA and a luminometric detection method was applied to the ELISA. Using this method native bovine IL-4 was detected in supernatants of PBMC stimulated with mitogens. In addition, high level secretion of IL-4 by Fasciola hepatica specific Th2 clones, but not by a Babesia bovis specific Th1 clone, was confirmed. The ELISA was also able to detect recombinant ovine IL-4. The pair of mAb used for ELISA could also be used for the detection of IL-4 spot forming cells by ELISPOT. In addition intracytoplasmic expression of IL-4 could be detected. The ability to detect ruminant IL-4 by three methods: ELISA, ELISPOT and by flow cytometric analysis of intracytoplasmic expression will permit studies of the role of this important cytokine in the immunology and pathogenesis of animal diseases.

Immunostimulatory CpG-modified plasmid DNA enhances IL-12, TNF-alpha, and NO production by bovine macrophages.

The immunogenicity of DNA vaccines is partially attributable to the adjuvant properties of bacterial plasmid DNA (pDNA) for B lymphocytes and professional antigen-presenting cells. In mice, modification of immunostimulatory sequences (ISSs), including CpG motifs, in pDNA vectors or oligodeoxynucleotides can increase or decrease their adjuvant properties. ISSs that stimulate optimal responses reportedly differ for murine and human leukocytes. We have previously characterized the mitogenic properties of oligodeoxynucleotides containing one AACGTT motif for bovine B lymphocytes. We now define cytokine responses by macrophages stimulated with pDNA engineered to contain an ISS comprising two AACGTT motifs. Macrophages activated with CpG-modified pDNA secreted significantly more interleukin-12, tumor necrosis factor-alpha, and nitric oxide than macrophages stimulated with unmodified pDNA or modified pDNA that contained nucleotides scrambled to remove CpG motifs. Engineered CpG-pDNA or CpG-oligodeoxynucleotides should be useful as vaccines or adjuvants to promote the enhanced type 1 responses important for protection against intracellular pathogens.

Angiotensin-induced myocardial necrosis and renal failure in the rabbit: distribution of lesions and severity in relation to plasma angiotensin II concentration and arterial pressure.

Angiotensin II was infused into conscious rabbits at various doses from 0.001 to 0.5 microgram . kg-1 . min-1 for 24 h, and caused multifocal myocardial necrosis, renal tubular necrosis and acute renal failure. The myocardial necroses were found principally in the left ventricle; only at the highest doses of angiotensin II were right ventricular lesions present. The endocardium was not involved and no arterial or arteriolar lesions were seen. Mean arterial plasma angiotensin II concentration during angiotensin infusion was closely correlated with the increase in arterial pressure, the height of the plasma urea at the end of the infusion and the severity of the induced myocardial lesions. The myocardial necroses could be a consequence of the induced hypertension, or a direct effect of angiotensin II, or a combination of effects, although their predominance in the left ventricle suggests high systemic arterial pressure is an important factor. Cardiac lesions were observed with plasma angiotensin II concentrations only some 2 to 3 fold normal values; it is therefore possible that similar myocardial abnormalities might occur as a result of rises in endogenous renin, for example, in experimental or clinical renovascular hypertension.

Angiotensin II causes vascular hypertrophy in part by a non-pressor mechanism.

Angiotensin II, when given in low doses, raises blood pressure slowly. When tested in vitro on vascular smooth muscle cells, it has mitogenic and trophic effects; it is not known if it has these effects in vivo. Our purpose was to determine whether vascular hypertrophy develops during slow pressor infusion of angiotensin II and, if so, whether it is pressure induced. Three experiments were done in rats infused subcutaneously with angiotensin II (200 ng/kg/min) by minipump for 10-12 days. Experiment 1: Angiotensin II gradually raised systolic blood pressure (measured in the tail) from 143 +/- 2 to 208 +/- 8 mm Hg (mean +/- SEM), significantly suppressing plasma renin and increasing threefold (NS) plasma angiotensin II. There was no loss of peptide in the pump infusate when tested at the end of the experiment. Experiment 2: In the perfused mesenteric circulation, vasoconstrictor responses to norepinephrine, vasopressin, and KCl were enhanced in rats given a slow pressor infusion of angiotensin II, but sensitivity of responses was not altered. This combination of changes suggests that vascular hypertrophy develops during slow pressor infusion of angiotensin II. Experiment 3: Vessel myography was done after angiotensin II infusion with and without a pressor response. Angiotensin II raised systolic blood pressure, increased heart weight, and produced myographic changes of vascular hypertrophy in the mesenteric circulation, increasing media width, media cross-sectional area, and media/lumen ratio. Hydralazine given with angiotensin II prevented the rise of pressure and the cardiac effect but not the vascular changes. Two-way analysis of variance showed that angiotensin II significantly increased media width, media cross-sectional area, and media/lumen ratio, all independent of hydralazine. Thus, although hydralazine inhibits the pressor and cardiac effects of angiotensin II, suggesting a pressor mechanism for the cardiac change, it does not inhibit structural vascular change, which suggests that at least part of the effect has a non-pressor mechanism.

A new cloning vector and expression strategy for genes encoding proteins toxic to Escherichia coli.

Here, we describe a modification of a plasmid, pT7-7 [Tabor and Richardson, Proc. Natl. Acad. Sci. USA 262 (1985) 1074-1078], that allows expression of inserted genes from the phage T7 RNA polymerase promoter. The modification is designed to suppress readthrough transcription from cryptic promoters and start points on the plasmid, in order to reduce expression in the absence of T7 RNA polymerase and thus improve the vector for use in the expression of highly toxic gene products. This vector (pT7SC) was used to stably clone the POL3 gene (encoding DNA polymerase delta) of Saccharomyces cerevisiae, which destabilizes all other cloning and expression vectors tested. Previously described expression strategies proved ineffective in overexpressing the POL3 gene. A new strategy was developed which relies on induction by infection with mutant T7 phage. This system efficiently overproduced the POL3 gene product.

Characterization of a cDNA encoding bovine interleukin 10: kinetics of expression in bovine lymphocytes.

A cDNA encoding bovine interleukin 10 (IL10) was cloned and sequenced using total cellular RNA derived from concanavalin A (ConA)-stimulated peripheral blood mononuclear cells (PBMC). A cDNA was produced with reverse transcriptase using oligo(dT) primers, and was amplified using primers chosen from consensus regions of the mouse and human IL10 genes. The nucleotide sequence derived from this cDNA shares 84, 79 and 78% homology with the human, mouse and rat cDNAs, respectively. The deduced amino-acid sequence shares an overall 77, 71 and 74% homology with the human, mouse and rat IL10 proteins, respectively. Northern blot analysis of the bovine IL10 mRNA reveals expression of a single 1.8-kb transcript, reaching maximal levels between 8 and 24 h, in ConA-stimulated peripheral T-cells, and weak expression in lipopolysaccharide-activated macrophages.

Cloning and expression of caprine interferon-gamma.

Caprine interferon-gamma (IFN-gamma) cDNA was cloned from mitogen stimulated peripheral blood mononuclear cell (PBMC) RNA utilizing the reverse transcription-polymerase chain reaction (RT-PCR). The cDNA open reading frame (ORF) is 498bp, encoding a putative 166 amino acid (aa) protein (19327Da). The predicted aa sequence homology of caprine IFN-gamma and the corresponding ovine, bovine and cervine cytokine is 98.8%, 95.2% and 92.8%, respectively. IFN-gamma cDNA was subcloned and expressed in two different plasmids under the control of either the human cytomegalovirus (CMV) immediate early promoter or the caprine arthritis-encephalitis virus long terminal repeat (CAEV LTR). Recombinant caprine IFN-gamma (rCaIFN-gamma) secreted by transfected COS-7 cells shared at least two antigenic epitopes with recombinant bovine IFN-gamma (rBoIFN-gamma) and exhibited biological activity in the vesicular stomatitis virus (VSV) cytopathic effect reduction assay. In-vivo expression of IFN-gamma cDNA promoted by the CAEV LTR was confirmed by the intramuscular (IM) injection of Balb/C mice with plasmid followed by Western blot analysis of mouse serum against purified rCaIFN-gamma produced in E. coli.

Antigenic variation in the persistence and transmission of the ehrlichia Anaplasma marginale.

Tick-borne transmission of ehrlichial pathogens requires rickettsemic reservoir hosts to maintain a population of infected vectors. Persistence in their respective mammalian hosts appears to be a common feature of the tick-transmitted ehrlichiae. How infection persists in immunocompetent hosts is unknown. In this review, we describe studies on Anaplasma marginale, an ehrlichial pathogen of cattle, that support antigenic variation as a primary mechanism of persistence.

Comparative effects of interleukin-12 and interleukin-4 on cytokine responses by antigen-stimulated memory CD4+ T cells of cattle: IL-12 enhances IFN-gamma production, whereas IL-4 has marginal effects on cytokine expression.

Interleukin-12 (IL-12) and IL-4 are important immunoregulatory cytokines that determine the fate of naive T cells during antigen priming in mice and also influence cytokine synthesis by differentiated murine and human T cells. The roles of these cytokines in regulating the differentiation and effector function of bovine T cells are less well studied. We investigated the ability of human IL-12 and bovine IL-4 to modify cytokine expression by antigen-stimulated T cells from cattle immune to the protozoal parasites Babesia bovis and Babesia bigemina or reactive with Mycobacterium bovis purified protein derivative. Peripheral blood mononuclear cells (PBMC) were cultured with specific antigen and IL-4 or IL-12 for 1 week. Then viable lymphoblasts consisting of predominantly CD4+ T cells were restimulated with antigen and antigen-presenting cells (APC) with or without cytokine. Cell lines were cultured for several weeks, and following restimulation with antigen and APC in the absence of exogenous cytokine, the cell lines were analyzed for proliferation, interferon-gamma (IFN-gamma) production, and expression of IL-2, IL4-, IL-10, or IFN-gamma transcript levels using a quantitative competitive RT-PCR. IL-12 and IL-4 had no effect on the composition of CD4, CD8, or gammadelta T cells in the cell lines or on the level of antigen-induced proliferation. IL-12 stimulated enhanced levels of IFN-gamma protein and transcript expression in all cell lines, with no consistent effects on IL-2 or IL-4 expression. In two B. bovis-specific cell lines, IL-12 suppressed IL-10 expression. IL-4 had no consistent effect on expression of any cytokine. These results indicate the use of IL-12 as an adjuvant to enhance type 1 cytokine responses in cattle during antigen priming.

Cloning of a cDNA encoding bovine interleukin-18 and analysis of IL-18 expression in macrophages and its IFN-gamma-inducing activity.

Interleukin-18 (IL-18) is a recently described cytokine that enhances interferon-gamma (IFN-gamma) production, either independently or synergistically with IL-12. These properties identify IL-18 as an immunoregulatory cytokine that may be pivotal in host defense against intracellular pathogens. We have isolated and sequenced a cDNA encoding bovine IL-18. The open reading frame (ORF) is 582 bp in length, encoding a predicted 192 amino acid (aa) precursor protein. Multiple sequence alignment demonstrated that bovine IL-18 has 65% and 78% identity with the predicted amino acid sequences of murine and human IL-18, respectively. IL-18 mRNA was constitutively present in bovine peripheral blood monocyte-derived macrophages (MDM), with no upregulation on stimulation with lipopolysaccharide (LPS). IL-18 transcripts were weakly detected in B lymphocytes but inducible in the B cell line BL-3. Human recombinant IL-18 (rHuIL-18) induced IFN-gamma production by PHA-stimulated peripheral blood mononuclear cells (PBMC), which was potentiated by rHuIL-12. Further, rHuIL-12 and rHuIL-18 enhanced proliferation of untreated PBMC. Antigen-specific T cell lines demonstrated IL-18-dependent enhancement of IFN-gamma production, indicating that bovine T cells are one of the leukocyte subsets that respond to IL-18. Analysis of IL-18 expression and its ability to induce IFN-gamma production by bovine lymphocytes are important considerations for understanding mechanisms of protective immunity and designing vaccines for intracellular pathogens.

Immunoregulatory roles of interleukin-13 in cattle.

Interleukin-13 (IL-13) is produced predominantly by helper T lymphocytes of the Th2 phenotype and mediates its effects on several immune cells, including B lymphocytes and macrophages, stimulating their proliferation, differentiation, and effector functions. IL-13 activates human B cells but has no detectable activity on murine B lymphocytes, suggesting that the activity of IL-13 varies among species. Our studies show that IL-13 enhances proliferation and differentiation of bovine B cells and upregulates cell surface major histocompatibility complex (MHC) class II expression. We examined mRNA expression of the putative signaling component of the bovine IL-13Ralpha1 homolog in several peripheral blood populations. After stimulation with calcium ionophore and phorbol ester, IL-13Ralpha1 mRNA levels appeared to be downmodulated in T cells, upregulated in macrophages and B cells, and unchanged in neutrophils. Together, these studies begin to provide insight into the relative importance of IL-13 in immunoregulation in cattle.

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.

Trophoblast IFN-tau differentially induces lymphopenia and neutropenia in lambs.

Type I interferons (IFN), including IFN-alpha and IFN-beta, cause severe lymphopenia, resulting from altered lymphocyte recirculation and redistribution. IFN-tau, a product of trophectoderm of ruminant conceptuses and new member of the type I IFN family has not been examined for its effect on leukocyte recirculation. Additionally, differential effects of type I IFNs on the redistribution and recirculation of subsets of T cells have not been reported. The present study determined the effects of IFN-tau on the redistribution and recirculation of ovine leukocytes and T cell subsets. Total peripheral blood leukocytes, lymphocytes, and segmented neutrophils were reduced (p < 0.05) following treatment of lambs with IFN-tau. Furthermore, administration of IFN-tau caused an acute, differential reduction in peripheral blood CD4+ T cells (p < 0.05), CD5+ cells (p < 0.05), and gammadelta TCR+ (p < 0.01) T cells but had no effect on CD8+ T cells (p > 0.05). IFN-tau reduced the percentage of gammadelta T cells by 8-fold and that of CD4+ T cells and CD5+ cells by <2-fold in peripheral blood when compared with control lambs. The reduction in leukocytes, lymphocytes, and neutrophils was observed as early as 6-12 h after administration of IFN-tau, but levels returned to control values within 48 h. These results indicate that IFN-tau, like other members of the type I IFN family, can have immediate effects on leukocyte recirculation and redistribution. The present study is the first to demonstrate that IFN-tau differentially regulates T cell recirculation with the greatest effect on gammadelta TcR+ T cells.

Upregulation of interleukin-4 and IFN-gamma expression by IFN-tau, a member of the type I IFN family.

Trophoblast interferon-tau (IFN-tau) is a new member of the type I IFN family that is produced in large quantities by the ruminant conceptus. Like other type I IFN, IFN-tau inhibits viral replication and activates natural killer (NK)-mediated cytotoxicity. In mice and humans, type I IFN enhances type 1 T helper (Th) cell responses, but the effects of type I IFN, including IFN-tau, on cytokine expression by bovine Th cells have not been described. The present study determined the effects of IFN-tau on interleukin-4 (IL-4), IFN-gamma, and IL-10 expression by antigen-specific, CD4+ T cell lines derived from cattle immune to either Babesia bovis, Babesia bigemina rhoptry-associated protein-1, or Anaplasma marginale. IFN-tau upregulated IFN-gamma secretion and steady-state levels of IFN-gamma and IL-4 mRNA by cell lines cultured for 3-6 weeks. In contrast, the steady-state levels of IL-10 mRNA were either not changed or inhibited at these times. Similar effects were obtained with human IFN-alpha. Comparison of the quantities of IFN-gamma, IL-4, and IL-10 transcripts in IFN-tau-treated or IFN-alpha-treated cultures revealed that even though IFN-gamma was the predominant cytokine expressed by all T cell lines, both IFN-gamma and IL-4 steady-state transcript levels were upregulated by a comparable degree. Thus, these studies demonstrate that IFN-tau is an immunomodulatory cytokine that promotes enhanced IL-4 and IFN-gamma responses by effector T cells but not, strictly speaking, Thl-biased responses in cattle. These results indicate the potential use of this cytokine as an adjuvant in ruminants to boost cell-mediated immune responses.

Induction of interleukin-6 and interleukin-12 in bovine B lymphocytes, monocytes, and macrophages by a CpG oligodeoxynucleotide (ODN 2059) containing the GTCGTT motif.

Bacterial DNA and synthetic oligodeoxynucleotides (ODN) that contain unmethylated CpG dinucleotides flanked by certain bases (CpG ODN) have been shown to activate murine and human B cells and to induce proinflammatory cytokines by monocytes/macrophages and dendritic cells (DC). However, the CpG ODN sequences optimal for mice and humans are different. In the current study, the effects of CpG ODN, which were defined to stimulate strong responses in either mouse or human leukocytes, were compared for stimulation of bovine B lymphocyte proliferation and macrophage cytokine mRNA expression. The optimal CpG ODN was then tested for induction of cytokines in peripheral blood mononuclear cells (PBMC) and purified B lymphocytes, monocytes, and macrophages. At a high ODN concentration (40 microM), all but two CpG ODN tested stimulated B cell proliferation, which was dependent on unmethylated CpG motifs. CpG ODN 2059 containing the GTCGTT motif shown to activate human leukocytes also promoted the highest level of bovine B cell proliferation at a lower concentration (10 microM) when compared with CpG ODN containing AACGTT or GACGTT motifs active for murine leukocytes. Furthermore, ODN 2059 induced interleukin-6 (IL-6) production by B lymphocytes and IL-6 and IL-12 production by PBMC, monocytes, and macrophages. In contrast, IL-1beta and tumor necrosis factor-alpha (TNF-alpha) production was either very low or undetectable. Consistent with increased IL-12 production, ODN 2059 also stimulated interferon-gamma (IFN-gamma) production by PBMC. Importantly, the levels of cytokines induced by ODN 2059 were comparable to those generated in response to Escherichia coli DNA. The weak TNF-alpha response combined with the vigorous IL-6 and IL-12 response to ODN 2059 indicate the potential use of this CpG ODN as an adjuvant to enhance both antibody-mediated and IFN-gamma-mediated macrophage activation, which are important for protection against disease caused by intracellular pathogens of cattle.

Immunization with Babesia bigemina rhoptry-associated protein 1 induces a type 1 cytokine response.

Rhoptry-associated protein-1 (RAP-1) homologues of Babesia bigemina and Babesia bovis are promising candidates for inclusion in subunit vaccines against these hemoprotozoan parasites. Partial protection against challenge infection has been achieved with native forms of these antigens, but the mechanism of immunity has not been thoroughly defined. We previously demonstrated that a panel of antigen-specific T helper cell clones derived from B. bigemina RAP-1-immunized cattle expressed relatively high levels of interferon-gamma (IFN-gamma) protein and transcript and low levels of interleukin-4 (IL-4), indicative of a type 1 immune response. In the current study we present evidence that subcutaneous immunization with native B. bigemina RAP-1 protein in RIBI adjuvant induces a predominant type 1 immune response in vivo, characterized by relatively high levels of IFN-gamma and IL-2 and low levels of IL-4 and IL-10 mRNA in the draining prescapular lymph node. Ex vivo restimulation of draining lymph node lymphocytes with specific antigen resulted in proliferation and enhanced expression of IL-2 and IFN-gamma, whereas IL-4 and IL-10 transcript levels remained relatively low. These findings show that our previously described cytokine profiles of antigen-specific cloned T cell lines are representative of autologous in vivo responses and confirm that type 1 recall responses to B. bigemina RAP-1 can be evoked in immunized animals by native parasite antigen.