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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., (© 2017 The Society for Applied Microbiology.)

Selenoprotein P (SePP) is a unique selenium-containing protein responsible for the transport and distribution of the essential trace element selenium (Se) through the human body with the concentration of SePP in human blood representing the most useful marker of Se nutritional status. Although SePP has been extensively studied, the structure of SePP in human plasma remains unresolved. Two potential isoforms of SePP have been identified by Western blot analyses distinguished principally by differences in migration (51 kDa and 61 kDa). The biological relevance of the smaller isoform has been called into question by several studies reporting only one major SePP form (69 kDa) suggesting that the shorter 51 kDa is an artifact of protease activity during the SePP purification process. A deficiency of these Western blot analyses is that no information can be gleaned regarding the Se content of the potential isoforms. This study reports a characterization of SePP isoforms in a human plasma Standard Reference Material representative of a healthy US population. Following immunoprecipitation, three SePP isoforms were unequivocally identified at 45 kDa, 49 kDa and 57 kDa (termed as SePP45, SePP49 and SePP57) by LC-MS/MS analyses from a spectral searching approach. Selenium (Se) was detected by gel electrophoresis LA-ICP-MS in SePP49 and SePP57 which was confirmed by the identification of three selenopeptides covering the SePP sequence from residues 312-346 by LC-MS/MS analyses utilizing a sequence searching approach. Conversely, neither Se nor peptides covering SePP sequence from residues 306-346 was identified in SePP45 which suggests that SePP45 is a truncated isoform transcriptionally terminated at the 2nd in-frame UGA codon thereby terminating the protein with the Ser residue at position 299. An additional band at 23 kDa was found to contain Se but no peptides of SePP. Instead, glutathione peroxidase 3 (GPx3) was unequivocally identified within the band presumably being co-immunoprecipitated with the SePP providing preliminary evidence that SePP and GPx3 interaction may take place in vivo.

Although many effects of staphylococcal superantigens (SAg) on T cells are well established, less is known about their effects on APC. In this study, bovine PBMC were stimulated with a low dose of staphylococcal enterotoxin C1 (SEC1). The phenotype of adherent cells (Ac) derived from bovine PBMC cultured with SEC1 [SEC1-stimulated Ac (sAc)] for 192 h was CD14(-), CD68(-), CD163(-), dendritic cell (DC)-specific ICAM-3-grabbing nonintegrin(+), MHC class II (MHC II)(high), CD11a(low), CD11b(high), CD11c(high), and CD1b(high), suggesting these cells were dendritic cells (DC). SEC1 also induced transcription of the CXCL1, -2, and -3 family, CXCL6, CCL2, and CCL5 genes in sAc, which increased rapidly but returned to basal levels by 48 h. In contrast, increased transcription of CCL3, CCL8, and CXCL12, responsible for mononuclear cell migration and chronic inflammation, was sustained. In vitro cell migration assays showed vigorous migration of granulocytes, followed by migration of mononuclear cells. The autologous MLR showed that sAc induced a dose-dependent proliferation of CD4(+) T cells and an even stronger proliferation of CD8(+) T cells. This effect was inhibited or reduced by pretreatment with mAb to CD11b, MHC II, or MHC II plus CD18. These results indicate that stimulation of bovine PBMC by SAg induces differentiation of monocytes into DC.

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.

We studied the tissue distribution of CD6(+) lymphocytes and cells expressing the CD6 ligand (also known as activated leukocyte cell adhesion molecule CD166) in calves by immunohistochemistry using an anti-bovine CD6 monoclonal antibody (mAb), a human CD6 (huCD6)-immunoglobulin G1 fusion protein (huCD6-Ig), and an anti-human CD166 (anti-huCD166) mAb. The huCD6-Ig and anti-huCD166 mAb bound to the sympathetic and parasympathetic nerve fibers but not to myelinated nerve fibers in the spinal nerve. Studies with human tissue using the anti-huCD166 mAb yielded identical patterns of labeling. Dense accumulations of CD6(+) lymphocytes were present in areas of the thymuses and spleens of calves, in areas innervated by huCD6-Ig(+) nerves. The cDNAs encoding the bovine CD166 and CD6 were isolated from the sympathetic ganglion and spleen, respectively. Predicted amino acid residues that are important for human and mouse CD6-CD166 binding were also conserved in bovine CD6 and CD166. Bovine CD166 transcripts were detected by reverse transcriptase-PCR in all the tissues that bound huCD6-Ig. These results show that the bovine orthologue of CD166 was constitutively expressed in the autonomic nervous systems of cattle and suggest that CD6(+) lymphocytes adhere to CD166(+) autonomic nerve terminals via CD6.

The role of various effector T cell populations in the bovine immune response to Mycobacterium bovis infection is poorly understood. This is largely due to the difficulties associated with performing in vivo challenge studies in the natural host species. In this report, we utilized a fetal bovine-severe combined immunodeficient (SCID-bo) xenochimeric mouse model to study the protective role of two putative effector cell types, CD8+ T cells and a subpopulation of gamma/delta T cells that express WC-1, a member of the cysteine-rich scavenger receptor superfamily (CRSR). We demonstrate that CD8+ T cells play a key role in protection and contribute substantially to bovine IFN-gamma mRNA levels at 30 days post-infection. The role of WC-1 bearing cells to protection was less definitive but our results suggest that this population may play a pivotal role early in infection. Granuloma architecture was altered in anti-WC-1 (ILA29) but not anti-CD8 (ILA51) -treated animals, suggesting that this population may be involved in recruitment of various cell types to sites of infection.

Theileria annulata is a tick-transmitted protozoan parasite of cattle, which transforms cells of macrophage (Mphi) or B cell lineage. Bone marrow cells, bone marrow cell-derived, and monocyte-derived Mphi were infected with T. annulata sporozoites, and the resulting cell lines were assessed for surface marker expression and function. Transformed lines expressed histocompatibility complex (MHC) class-I and II, CD44, CD45, and the myeloid marker DH598-surface markers CD14, CD11b, M-M7, TH57A, and to a lesser extent CD11a/CD18, CD11c, and ACT(B), were down-regulated. Likewise, transformed cells failed to express Mphi functions (Fc-receptor-mediated phagocytosis, phorbol myristate acetate-induced oxidative burst, lipopolysaccharide-induced tumor necrosis factor alpha, and nitric oxide generation and procoagulant activity up-regulation). Mphi origin was assured by homogeneity of the starting population, cloning of cells by limiting dilution, and repeated microscopic and flow cytometric monitoring of the cell lines. Elimination of the parasite by treatment with BW720c resulted in the re-acquisition of monocyte lineage properties, as evidenced by up-regulation of CD14, and by re-acquisition of the capacity to ingest opsonized sheep red blood cells and bacteria. Thus, Mphi transformed by T. annulata appear to undergo a process of parasite-induced dedifferentiation but reassume the differentiated phenotype upon elimination of the parasite.

Studies were conducted to determine the reactivity of six monoclonal antibodies specific for major histocompatibility complex class II molecules expressed on a bovine B cell line homozygous for BoLA-DR and BoLA-DQ alleles. Direct immunoprecipitation, serial immunodepletion experiments, and two-dimensional gel electrophoresis revealed that these antibodies reacted with three different molecules. Bovine orthologues of HLA-DR were recognized by three monoclonal antibodies--H34A, TH12A, and TH14B. Orthologues of HLA-DQ were characterized by two other monoclonal antibodies, TH22A and TH81A. A third BoLA class II antigen, neither DR nor DQ was revealed by the last monoclonal antibody H42A. The relation of this molecule to known molecules in humans and other species remains to be established. However, cumulative data suggest that the determinant is expressed on a molecule related to HLA-DP.

A panel of eight murine monoclonal antibodies (MAbs) were produced against heat-killed Escherichia coli J5 and shown to react with J5 lipopolysaccharide (LPS) by enzyme-linked immunosorbent assay (ELISA). These antibodies were then assayed by a suspension ELISA for reactivity with up to 20 heterologous smooth or rough isolates of gram-negative bacteria, which were assayed after heat or formalin treatment, or as live cells. Extracted LPS from the same bacteria were tested for reactivity with the MAbs by direct ELISA. The MAbs demonstrated broad cross-reactivity with most heat-treated bacteria. In contrast, cross-reactivity of the MAbs with live or formalin-treated bacteria was limited almost exclusively to E. coli J5, Hemophilus species, or rough mutants of Salmonella minnesota. Reactivity with extracted LPSs and lipid A varied considerably depending on the MAb. Further, when Western blotting was used as the assay only four of eight MAbs reacted with J5 LPS, and none of the MAbs reacted with LPS from smooth S. minnesota or any of its rough mutants. Adsorption of the MAbs with acid hydrolyzed, boiled, or live E. coli J5 prior to ELISA of the MAbs with J5 LPS supported evidence that none of the MAbs were specific for lipid A and that reactivity was greater with boiled than with live cells. Thus, the cross-reactivity of antibodies to E. coli J5 LPS is dependent on the physical state of the bacteria or LPS used for assay, the assay used, and the specificity of the antibody.

The levels of IgM, IgY, and IgA and the development of specific antibody to Marek's disease virus (MDV) and sheep red blood cells (SRBC) in young chickens susceptible and resistant to Marek's disease were compared after exposure to MDV. No significant difference was noted in the immunoglobulin levels. However, the antibody response to MDV and SRBC occurred more rapidly in susceptible birds. The initial titer of antibody to these antigens was higher. These differences in response, however, were transient. At 3 weeks post exposure, the levels of IgM antibodies to MDV and antibodies to SRBC were similar in the two lines of chickens. At 6 weeks, the levels of IgY antibodies to MDV and antibodies detected by the agar gel precipitation test were similar.