Investigating the feasibility of ICP-MS/MS for differentiating NIST salmon reference materials through determination of Sr and S isotope ratios.

Inductively coupled plasma tandem mass spectrometry (ICP-MS/MS) was investigated for possible use in food fraud studies through the measurement of strontium and sulfur isotope ratios. Oxygen mass shift mode was applied to shift 87Sr/86Sr and 34S/32S isotope ratios to their respective oxides, 87Sr16O+/86Sr16O+ and 34S16O+/32S16O+, effecting a gas-phase chemical separation of the elements from Rb and Kr (for Sr) and molecular N and O species, along with P- and S-hydrides (for S). A total least squares regression approach was employed to generate the isotope ratio data from time-resolved analyses, and method uncertainties and accuracies were determined. The utility of the approach was shown by using the Sr and S isotope ratios together to differentiate between NIST RM 8256 Wild-Caught Coho Salmon and NIST RM 8257 Aquacultured Coho Salmon. These materials are currently under development at NIST as certified food fraud standards and method evaluation materials for comprehensive chemical analysis.

Immune cell populations residing in mesenteric adipose depots and mesenteric lymph nodes of lean dairy cows.

Inconsistent evidence of inflammatory immune cell infiltrates in adipose tissues with extensive triglyceride mobilization raises the possibility that regulatory or anti-inflammatory immune cell populations reside within the mesenteric adipose tissue (MAT) and mesenteric lymph nodes (MLN). These resident immune cell populations may be involved in attenuating the inflammatory response. We explored the immune cell population of MAT and MLN collected from lean, lactating Holstein cows without apparent disease in an abattoir (n = 42). Lean cows had a body condition score of 2.6 ± 0.1 (mean ± SD) with a greater frequency of adipocyte area occurring in small rather than large adipocytes. Cells were labeled with monoclonal antibodies specific to bovine leukocyte antigens for enumeration by flow cytometry. Within both lymph node and adipose tissues, relatively large subpopulations of cells expressed the ß2 integrins CD11b and CD11c, class II major histocompatibility antigens (MHCII), and the SIIRP-1α receptor (CD172a) typical of dendritic cells and macrophages. Macrophage/dendritic cell heterogeneity was marked by ß2 integrin expression alone or in conjunction with CD172a or MHCII across subpopulations from both tissues; CD209, the DC-SIGN c-type lectin receptor of dendritic cells, was not detected by fluorescence-activated cell sorting in either tissue. Lymphocytes comprised 74.1 ± 3.7% and 13.7 ± 3.7% of the MLN and MAT cell populations, respectively, and CD3+CD4+ lymphocytes accounted for 49.8 ± 9.9% of the MLN and 6.13 ± 1.23% of the MAT cells. Fox P3+ regulatory lymphocytes comprised 15.3 ± 1.1% and 6.73 ± 0.52% of the MLN and MAT cells, whereas γδ+ lymphocytes accounted for 6.65 ± 0.74% and 3.91 ± 0.43% of the MLN and MAT cells, respectively. Subpopulations of CD3+CD8+ cytotoxic T cells and CD3+CD11c+ innate lymphocytes were present in MLN but not MAT. These results show that subpopulations of resident tissue macrophages, dendritic cells, T helper lymphocytes, regulatory T lymphocytes (Tregs), and γδ lymphocytes reside in mesenteric lymph nodes and adipose tissues. Balance in the innate and adaptive immune functions embedded in these tissues could support metabolic health.

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.

Prevalence of Bovine Tuberculosis in Egyptian Cattle and the Standardization of the Interferon-gamma Assay as an Ancillary Test.

Bovine tuberculosis (bTB) caused primarily by Mycobacterium bovis continues to cause significant losses in the cattle industry and is a major public health problem. Despite its worldwide application, the IFN-γ assay has not been applied in Egypt. The aim of this study was to determine the appropriate cut-off value of IFN-γ assay to complement the skin test screening in Egypt. The relative sensitivity (Ser ) of PPD and antigen cocktail-based IFN-γ assays (IFN-γ-BA and IFN-γ-EC) was analysed retrospectively, relative to bTB confirmatory tests (culture and PCR), using single cervical tuberculin (SCT) test reactors during 2011-2013. The absolute specificity (Sp) was studied using blood samples collected from cattle from one bTB-free herd. Analysis of the bTB database-generated sheets indicates the infection rate had decreased from 2009 to 2012 and then increased in 2013. The disease is concentrated in the Egyptian Nile Delta and Valley relative to elsewhere in the country. The cut-offs for IFN-γ-EC assay could be optimized to provide higher sensitivity, comparable to cut-offs for IFN-γ-BA assay. Data analysis suggests (PPDbOD  > 0.1, PPDbOD  - NILOD  > 0.05 and PPDbOD  > PPDaOD ) and (ECOD  - NILOD  ≥ 0.1) cut-off strategies to get optimal IFN-γ-BA and IFN-γ-EC assays results respectively. To our knowledge, this is the first report describing the prevalence of bTB in cattle in Egypt and pointing out the appropriate cut-off criteria to optimize IFN-γ assay as a routine ancillary test for diagnosis of bTB in Egypt.

Contributions of conventional and heavy-chain IgG to immunity in fetal, neonatal, and adult alpacas.

In addition to conventional immunoglobulins, camelids produce antibodies that do not incorporate light chains into their structures. These so-called heavy-chain (HC) antibodies have incited great interest in the biomedical community, as they have considerable potential for biotechnological and therapeutic application. Recently, we have begun to elucidate the immunological functions of HC antibodies, yet little is known about their significance in maternal immunity or about the B lymphocytes that produce them. This study describes the application of isotype-specific reagents toward physiological assessments of camelid IgGs and the B cells that produce them. We document the specificities of monoclonal antibodies that distinguish two conventional IgG1 isotypes and two HC IgG3 variants produced by alpacas. Next, we report that the relative concentrations of five isotypes are similar in serum, milk, and colostrum; however, following passive transfer, the concentrations of HC IgG2 and IgG3 declined more rapidly than the concentration of conventional IgG1 in the sera of neonates. Finally, we assessed the distribution of B cells of distinct isotypes within lymphoid tissues during fetal and adult life. We detected IgG1, IgG2, and IgG3 in lymphocytes located in lymph node follicles, suggesting that HC B cells affinity mature and/or class switch. One IgG3 isotype was present in B cells located in ileal Peyer's patches, and one conventional IgG1 isotype was detected in splenic marginal zone B cells. Our findings contribute to the growing body of knowledge pertaining to HC antibodies and are compatible with functional specialization among conventional and HC IgGs in the alpaca.

The influence of age and Rhodococcus equi infection on CD1 expression by equine antigen presenting cells

There is a distinct age-associated susceptibility of horses to Rhodococcus equi infection. Initial infection is thought to occur in the neonatal and perinatal period, and only foals less than 6 months of age are typically affected. R. equi is closely related and structurally similar to Mycobacterium tuberculosis, and causes similar pathologic lesions. Protective immune responses to M. tuberculosis involve classical major histocompatibility complex (MHC)-restricted T cells that recognize peptide antigen, as well as MHC-independent T cells that recognize mycobacterial lipid antigen presented by CD1 molecules. Given the structural similarity between these two pathogens and our previous observations regarding R. equi-specific, MHC-unrestricted cytotoxic T lymphocytes (CTL), we developed 3 related hypotheses: (1) CD1 molecules are expressed on equine antigen presenting cells (APC), (2) CD1 expression on APC is less in foals compared to adults and (3) infection with live virulent R. equi induces up-regulation of CD1 on both adult and perinatal APC. CD1 expression was examined by flow cytometric analysis using a panel of monoclonal CD1 antibodies with different species and isoform specificities. RESULTS: Three CD1 antibodies specific for CD1b showed consistent cross reactivity with both foal and adult monocyte-derived macrophages (MDM). CD1b and MHC class II expression were significantly higher on adult MDM compared with foals. R. equi infected MDM showed significantly lower expression of CD1b, suggesting that infection with this bacterium induces down-regulation of CD1b on the cell surface. Histograms from dual antibody staining of peripheral blood mononuclear cells also revealed that 45-71% of the monocyte population stained positive for CD1b, and that the majority of these also co-expressed MHC II molecules, indicating that they were APC. The anti-CD1 antibodies showed no binding or minimal binding to bronchoalveolar lavage (BAL)-derived macrophages.

Development of monoclonal antibodies to detect bovine FOXP3 in PBMCs exposed to a staphylococcal superantigen.

The role of regulatory T cells (Tregs) is well documented in immune homeostasis and protection against autoimmune disease. Forkhead box protein 3 (FOXP3) has been shown to be essential for the development and function of T(reg). Due to the lack of tools for FOXP3 detection in certain species, understanding the role of Treg in a variety of ruminant diseases has been hampered. In this study, we developed monoclonal antibodies (mAbs) against bovine FOXP3 using recombinant bovine FOXP3 lacking the forkhead domain as an immunogen. The specificity of the mAbs was confirmed by immunoblot and mass spectrometry. Expression of FOXP3 was induced in bovine PBMCs after 6 d of exposure to staphylococcal enterotoxin type C1 (SEC1) in vitro. Similar to findings in mice and humans, expression of FOXP3 was restricted to CD4+ CD25+ T cells. Transcriptional analysis of bovine TCR variable regions of the beta chain (boVbeta) showed that transcription of boVbeta sequences reactive with SEC1 increased for 6 d, and then boVbeta sequences non-reactive with SEC1 rapidly increased in the cultures. This indicates that induction of FOXP3+ CD4+ CD25+ Tregs by SEC1 is not Vbeta restricted. The FOXP3 mAbs developed in this study will be useful in the further investigation of the role of Treg in staphylococcal pathogenesis in bovine mastitis and other ruminant diseases.

Myocarditis associated with foot-and-mouth disease virus type O in lambs.

The present study describes the pathogenetic mechanisms of myocarditis in 9 lambs that died in a foot-and-mouth disease outbreak in Samsun, Turkey. In all the heart samples tested, ELISA and sequencing for phylogenetic analyses showed that the virus, namely O/TUR/Samsun/05, was associated with the PanAsia pandemic strain of foot-and-mouth disease virus (FMDV) type O. The lambs had myocardial lesions but no typical vesicular lesions. In situ reverse transcription showed that many cardiomyocytes and some interstitial cells were positive for FMDV type O. Inflammatory infiltration, hyaline degeneration, and necrosis of sheets of myocytes were observed. The cellular infiltrates were mononuclear cells, including many lymphocytes, macrophages, a few plasma cells, and neutrophils. Major histocompatibility complex Class II+ dendritic and mononuclear cells, gammadelta T cells, CD172A+ and CD14+ macrophages and monocytes, and IgM+ B cells were detected mainly in the infected hearts. Inducible nitric oxide synthetase (iNOS) was seen mostly in areas of inflammation infiltrated by large numbers of cells. Of the 2 alpha-subunits of integrin known to be used as receptors by FMDV in epithelial tissues, CD49e (integrin alpha5) was detected in the membranes of cardiac myocytes with intercalated discs, but CD51 (integrin alphaV) was not detected in cardiac myocytes from infected or normal lambs. Interstitial and inflammatory cells were positive for both integrin subunits. The terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling (TUNEL)-positive signal was detected in the nuclei of both cardiac myocytes and interstitial cells from infected lambs. These findings suggest that the iNOS expressed by inflammatory cells in lesions may have a deleterious effect on cardiac myocytes in these lesions.

Immunohistochemical study of experimental malignant catarrhal fever in rabbits.

Malignant catarrhal fever (MCF) is an often-fatal lymphoproliferative disease of a variety of ungulates that occurs worldwide. It is caused by either of the highly related but distinct gammaherpesviruses alcelaphine herpesvirus-1 (AlHV-1, wildebeest reservoir) or ovine herpesvirus-2 (OvHV-2, sheep reservoir). MCF in rabbits is an excellent model as it closely resembles the disease in susceptible ungulates that include cattle, deer and bison. In this study, newly available and previously characterized monoclonal antibodies specific for rabbit leucocyte differentiation molecules were used to perform a detailed immunohistochemical examination of both AlHV-1 MCF and OvHV-2 MCF in rabbits. Differences in the MCF caused by the two viruses included: less tissue necrosis and more lymphoid cell accumulations in AlHV-1 MCF compared with OvHV-2 MCF, and in particular marked tissue necrosis in the mesenteric lymph node, appendix and liver of OvHV-2-infected animals when compared with either other tissues in OvHV-2 MCF or AlHV-1 MCF lesions in any tissue. In both AlHV-1 MCF and OvHV-2 MCF, lymphoid cell accumulations in lymphoid and non-lymphoid tissues consisted mainly of T-cells with a corresponding absence of B-cells. CD8(+) T-cells accounted for a proportion of these in the non-lymphoid tissues, but there was evidence for the accumulation of an unidentified T-cell subset/subsets as well. This study extends our understanding of the mechanisms of immuno-pathogenesis of MCF.

Lymphocyte subset distribution in apparently normal and single intradermal test-positive water buffaloes analyzed by flow cytometry.

Monoclonal antibodies (mAbs) against bovine lymphocyte cell surface antigens namely, MHC Class I, MHC class II (DP, DQ and DR), CD3, CD4, CD8, gamma delta TCR, WC1N1 and WC1N2, were tested for their reactivity on apparently normal buffalo mononuclear cells prepared from spleen, lymph nodes and peripheral blood. All the mAbs cross-reacted with the buffalo mononuclear cells. The mean (+/-SD) CD4:CD8 cell ratio in the peripheral blood of apparently normal buffaloes was 1.08+/-0.049 while in the spleen and lymph nodes it was 0.90+/-0.080 and 1.81+/-0.430, respectively. The lymphocyte subsets in the buffaloes positive for tuberculosis by the single intra dermal (SID) test was found to be altered; the CD4 cells were reduced while the CD8 and gamma delta cells were increased. The mean CD4:CD8 ratio in the SID positive buffaloes was 0.36+/-0.010.

Immunohistochemical evaluation of inflammatory infiltrate in the skin and lung of lambs naturally infected with sheeppox virus.

The present study describes immunophenotypic characteristics of inflammatory infiltrate in the skin and lung of lambs naturally infected with sheeppox virus (SPV). Three lambs revealed typical cutaneous and pulmonary lesions of sheeppox. Histologically, cutaneous and pulmonary lesions consisted of hyperplastic and/or degenerative changes in the epithelium with mononuclear cells, neutrophils, and typical sheeppox cells (SPCs), which had a vacuolated nucleus and marginated chromatin with occasional granular intracytoplasmic inclusions. The inflammatory infiltrate in pox lesions in both skin and lung was characterized by the presence of MHC II+ dendritic cells, CD4+, CD8+, gammadelta+ T cells, IgM+ cells, and CD21+ cells. Loss of expression of MHC I and MHC II antigens was observed in the affected areas of skin and lung. SPCs, stained with anti-SPV antibody, were also positive for CD14 and CD172A, antigens expressed on monocytes and macrophages. CD14 and CD172A negative SPCs were considered to be SPV infected degenerated epithelial cells or fibroblasts.

Establishment of Neospora caninum antigen-specific T cell lines of primarily CD4 T cells.

Neosporosis is an important cause of pregnancy loss in cattle worldwide. Protective immunity against Neospora caninum infection may include both cell-mediated (CMI) and humoral immune responses. This study was to establish short-term antigen-specific T cell lines composed of primarily CD4(+)T cells from peripheral blood lymphocytes (PBL) of infected cows, which may be used to identify immunodominant antigens for the development of N. caninum vaccines. Crude N. caninum tachyzoite antigen was prepared from in vitro derived N. caninum tachyzoites. Multiple T cell lines were established and maintained for 11 weeks by weekly re-stimulation with N. caninum antigen and antigen-presenting cells. All cell lines responded highly to antigen between weeks 3 and 11. Phenotypically, these cells were composed primarily of CD4(+)T cells between weeks 2-8, with a gradual expansion of gamma/delta(+)T cells thereafter. The results indicate that N. caninum-specific T cell lines can be established and maintained without exogenous T cell growth factors and may be used to identify N. caninum antigens. This research will enhance our understanding of bovine CMI to neosporosis and may facilitate development of a proven neosporosis vaccine.

Cell proliferation, apoptosis, and B- and T-lymphocytes in Peyer's patches of the ileum, in thymus and in lymph nodes of preterm calves, and in full-term calves at birth and on day 5 of life.

Peyer's patches, thymus, and lymph nodes contain the majority of lymphocytes. We have studied proliferation rates, apoptosis rates, and numbers of B- and T-lymphocytes in Peyer's patches in ileum, thymus, and mesenterial and prescapular lymph nodes (LM and LP) in unfed preterm calves (GrP; born 13 d before expected normal term after dams were injected with prostaglandin F2alpha and glucocorticoids) and normal-term calves (GrF) immediately after birth and on d 5 of life after feeding colostrum for 4 d (GrC). Immunohistochemical methods in conjunction with incorporation of 5-bromo-2'-deoxyuridine or terminal deoxynucleotidyl transferase-mediated X-dUTP nick end labeling were used to evaluate cell proliferation rates and apoptosis rates, respectively. The number of T- and B-lymphocytes was determined with monoclonal antibodies directed against CD3 and CD79, respectively. In GrF compared with GrP, there were higher numbers of proliferating and apoptotic cells in LM and LP, of B-lymphocytes in paracortex and follicles of LM and LP, and of proliferating cells in cortex and medulla of thymus. In thymus cortex and medulla, numbers of proliferating cells were higher in GrC than in GrF. Apoptotic rates were generally smaller at all sites of Peyer's patches in GrC than in GrF, and proliferation rates increased from GrP to GrF in intrafollicular areas and from GrF to GrC in all tissues. Numbers of T-lymphocytes in Peyer's patches were higher in GrF than in GrP, but lower in GrC than in GrF, except in the domes. Numbers of B-lymphocytes did not change in Peyer's patches despite high proliferation and low apoptotic rates, suggesting that they leave Peyer's patches during the first days of life. In conclusion, proliferation and apoptosis rates and numbers of B- and T- lymphocytes in Peyer's patches in ileum, thymus, and LM and LP exhibited different developmental changes and were affected by feeding.

Early induction of humoral and cellular immune responses during experimental Mycobacterium avium subsp. paratuberculosis infection of calves.

Johne's disease (paratuberculosis) of cattle is widespread and causes significant economic losses for producers due to decreased production and poor health of affected animals. The chronic nature of the disease and the lack of a reproducible model of infection hinder research efforts. In the present study, instillation of Mycobacterium avium subsp. paratuberculosis into the tonsillar crypts of neonatal calves resulted in peripheral colonization as detected by antemortem culture of feces and postmortem (320 days postchallenge) culture of intestinal tissues. Antigen-specific blastogenic, gamma interferon (IFN-gamma), and nitric oxide responses by blood mononuclear cells from infected calves exceeded prechallenge responses beginning 194 days postchallenge. Upon in vitro stimulation with paratuberculosis antigens, CD4(+) cells from infected calves proliferated, produced IFN-gamma, and increased expression of CD26 and CD45RO (indicative of an activated memory phenotype). Utilizing a lipoarabinomannan-based enzyme-linked immunosorbent assay, specific serum immunoglobulin was detected as early as 134 days postchallenge and generally increased after this time point. Two antigens of approximately 50 and approximately 60 kDa were particularly immunodominant early in infection, as shown by immunoblot with serum collected within 2 weeks postchallenge. Findings indicate that the intratonsillar inoculation route will prove useful as an experimental model for paratuberculosis infection. Additionally, this study confirms that mycobacteria-specific antibody is detectable early in the course of experimental Johne's disease, even preceding the development of specific cell-mediated responses.

Use of the mannan receptor to selectively target vaccine antigens for processing and antigen presentation through the MHC class I and class II pathways.

Extensive studies have shown that synthetic and recombinant vaccines developed against hemoparasites have not been as effective as whole parasites or crude membrane fractions in eliciting protective immunity. A possible reason is that synthetic vaccines are not being presented in a form that induces the appropriate immune response. We have developed a bovine model system to evaluate the ability of adjuvant compounds to induce an immune response to peptide antigens dominated by a cytokine profile with a Type 1 (cell-mediated) or Type 2 (humoral) bias. In the initial testing of this system, we found that mRNA expression of certain cytokines (interleukin [IL]-1beta, IL-6, IL-12, IL-15, GM-CSF, iNOS, and tumor necrosis factor [TNF]-alpha) is enhanced when monocyte-derived macrophages are stimulated with peptide antigen conjugated with mannan under oxidizing conditions compared to peptide conjugated with reduced mannan. The data suggest this model will be useful in identifying adjuvant systems that selectively modulate the cytokine profile of antigen presenting cells at the time of antigen presentation and the consequent downstream maturation of naive T cells to effector cells with Type 1 or Type 2 cytokine bias.

Age-related innate immune response in calves to Babesia bovis involves IL-12 induction and IL-10 modulation.

There is a strong innate immunity in calves to infection with Babesia bovis. Interleukin (IL)-12 and IL-10 have been shown in vitro to be important immunoregulatory cytokines. Here we demonstrate in vivo that the protective innate response in young calves to infection with virulent B. bovis involves the early appearance of IL-12 and interferon-gamma (IFN-gamma) transcripts in the spleen. In contrast, IL-12 and IFN-gamma mRNA expression in the spleens of adult cattle that succumbed to the infection was delayed and depressed and occurred within the context of IL-10 expression. Also in contrast with calves, there was no detectable antibody response before death in adults. A vigorous CD8+ T-cell expansion occurred in the spleens of both calves and adults.

Scavenger receptor cysteine-rich domains 9 and 11 of WC1 are receptors for the WC1 counter receptor.

Workshop cluster 1 (WC1) is a member of the scavenger receptor cysteine-rich (SRCR) superfamily that includes CD5, CD6, CD163, and M160. Bovine WC1 consists of 11 SRCR domains, a unique domain 1, and two homologous 5 SRCR domain cassettes, WC1 domains 2-6 and 7-11. The porcine orthologue of WC1 contains five SRCR domains with a different domain arrangement. Although the function of WC1 is unknown, WC1 is proposed to be an accessory or homing molecule. Thus, identification of cells that express the counter receptor for WC1 (WC1-CR) is critical to understanding the function of WC1. For this reason, we constructed WC1-human immunoglobulin G1 fusion proteins to identify the binding domain of WC1 and cells that express the WC1-CR. Immunohistochemical analysis revealed WC1 domains 9 and 11 bind cells with macrophage and dendritic cell morphology and cells in ellipsoids in the spleen. These results and the finding of conserved signaling motifs in the cytoplasmic tail suggest WC1 may be an accessory molecule.

Cloning of bovine CD69.

CD69 is rapidly inducible on various hematopoietic cells upon stimulation and is detectable as an early activation antigen. Although CD69 is well characterized in human and mouse, no information is available on bovine CD69. We report here that, bovine CD69 was cloned from a cDNA expression library prepared from activated peripheral blood lymphocytes. The full-length cDNA contained an 80bp 5' untranslated region, followed by a 600bp coding region and AU-rich motifs in a 3' untranslated region (GenBank accession number AF272828). Comparison of the bovine CD69 coding sequence reveals 69.4 and 78.2% nucleotide sequence identities with mouse and human CD69, respectively. The predicted amino acid sequence of bovine CD69 shares 56.3 and 62.3% sequence identity when compared with mouse and human CD69, respectively. Bovine CD69 has the highly conserved amino acid sequences found in the C-type lectin family, suggesting that the conserved residues may be important for conformation and binding to the, as yet unidentified ligand. In addition, the cytoplasmic tail of bovine CD69 has two casein kinase-2 (CK-2) phosphorylation sites. These data suggest that bovine CD69 plays an important role in the activation of lymphocytes.

IL-4 and IL-10 inhibition of IFN-gamma- and TNF-alpha-dependent nitric oxide production from bovine mononuclear phagocytes exposed to Babesia bovis merozoites.

The requirement for IFN-gamma and/or TNF-alpha as co-stimulants with Babesia bovis merozoites for nitric oxide (NO) production was examined, as well as the regulatory role of IL-4 and IL-10. Purified B. bovis merozoites did not induce the production of NO in undifferentiated monocytes without addition of exogenous IFN-gamma and TNF-alpha unless the monocytes taken ex vivo were producing TNF-alpha endogenously. Under the latter condition, the NO production resulting from merozoite stimulation remained IFN-gamma-dependent. There was no evidence for endogenous synthesis of TNF-alpha in monocyte-derived macrophages (MDM), and merozoites alone were incapable of inducing TNF-alpha mRNA in MDM. However, while merozoites plus IFN-gamma induced TNF-alpha mRNA expression in MDM, NO was not produced. Both IL-4 and IL-10 inhibited expression of iNOS and production of NO in merozoite-stimulated monocytes.

Identity of activation molecule 3 on superantigen-stimulated bovine cells is CD26.

Most CD8(+) T cells in cultures of bovine mononuclear cells stimulated with staphylococcal enterotoxin C1 develop an unusual phenotype characterized by expression of activation molecule 3 (ACT3). This superantigen-dependent phenotype may be relevant to immunopathogenesis mediated by certain microbial toxins. The size and N-terminal sequence of immunoprecipitated ACT3 indicate that ACT3 is the bovine orthologue of CD26.