Lindsay M. Fry

Characterization and use of new monoclonal antibodies to CD11c, CD14, and CD163 to analyze the phenotypic complexity of ruminant monocyte subsets.

The sequencing of the bovine genome and development of mass spectrometry, in conjunction with flow cytometry (FC), have afforded an opportunity to complete the characterization of the specificity of monoclonal antibodies (mAbs), only partially characterized during previous international workshops focused on antibody development for livestock (1991, Leukocyte Antigens in Cattle, Sheep, and Goats; 1993, Leukocyte Antigens of Cattle and Sheep; 1996, Third Workshop on Ruminant Leukocyte Antigens). The objective of this study was to complete the characterization of twelve mAbs incompletely characterized during the workshops that reacted with molecules predominantly expressed on bovine monocytes and use them to provide further information on the phenotypic complexity of monocyte subsets in ruminants. Analysis revealed that the mAbs could be grouped into three clusters that recognize three different molecules: CD11c, CD14, and CD163. Following characterization, comparison of the patterns of expression of CD14 and CD163 with expression of CD16, CD172a, and CD209 revealed the mononuclear cell population is comprised of multiple subsets with differential expression of these molecules. Further analysis revealed the epitopes recognized by mAbs to CD14 and CD163 are conserved on orthologues in sheep and goats. In contrast to CD14 that is also expressed on sheep and goat granulocytes, CD163 is a definitive marker for their monocytes.

Phenotype and Function of CD209+ Bovine Blood Dendritic Cells, Monocyte-Derived-Dendritic Cells and Monocyte-Derived Macrophages

Phylogenic comparisons of the mononuclear phagocyte system (MPS) of humans and mice demonstrate phenotypic divergence of dendritic cell (DC) subsets that play similar roles in innate and adaptive immunity. Although differing in phenotype, DC can be classified into four groups according to ontogeny and function: conventional DC (cDC1 and cDC2), plasmacytoid DC (pDC), and monocyte derived DC (MoDC). DC of Artiodactyla (pigs and ruminants) can also be sub-classified using this system, allowing direct functional and phenotypic comparison of MoDC and other DC subsets trafficking in blood (bDC). Because of the high volume of blood collections required to study DC, cattle offer the best opportunity to further our understanding of bDC and MoDC function in an outbred large animal species. As reported here, phenotyping DC using a monoclonal antibody (mAb) to CD209 revealed CD209 is expressed on the major myeloid population of DC present in blood and MoDC, providing a phenotypic link between these two subsets. Additionally, the present study demonstrates that CD209 is also expressed on monocyte derived macrophages (MoΦ). Functional analysis revealed each of these populations can take up and process antigens (Ags), present them to CD4 and CD8 T cells, and elicit a T-cell recall response. Thus, bDC, MoDC, and MoΦ pulsed with pathogens or candidate vaccine antigens can be used to study factors that modulate DC-driven T-cell priming and differentiation ex vivo.

Immunization-Induced Anaplasma marginale-Specific T-Lymphocyte Responses Impaired by A. marginale Infection Are Restored after Eliminating Infection with Tetracycline

ABSTRACT Infection of cattle with Anaplasma marginale fails to prime sustained effector/memory T-cell responses, and high bacterial load may induce antigen-specific CD4 T exhaustion and deletion. We tested the hypothesis that clearance of persistent infection restores the exhausted T-cell response. We show that infection-induced T-cell exhaustion, characterized as loss of antigen-specific proliferation, and gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) production are partially restored in cattle following clearance of persistent infection with tetracycline.

East Coast fever caused by Theileria parva is characterized by macrophage activation associated with vasculitis and respiratory failure

Respiratory failure and death in East Coast Fever (ECF), a clinical syndrome of African cattle caused by the apicomplexan parasite Theileria parva, has historically been attributed to pulmonary infiltration by infected lymphocytes. However, immunohistochemical staining of tissue from T. parva infected cattle revealed large numbers of CD3- and CD20-negative intralesional mononuclear cells. Due to this finding, we hypothesized that macrophages play an important role in Theileria parva disease pathogenesis. Data presented here demonstrates that terminal ECF in both Holstein and Boran cattle is largely due to multisystemic histiocytic responses and resultant tissue damage. Furthermore, the combination of these histologic changes with the clinical findings, including lymphadenopathy, prolonged pyrexia, multi-lineage leukopenia, and thrombocytopenia is consistent with macrophage activation syndrome. All animals that succumbed to infection exhibited lymphohistiocytic vasculitis of small to medium caliber blood and lymphatic vessels. In pulmonary, lymphoid, splenic and hepatic tissues from Holstein cattle, the majority of intralesional macrophages were positive for CD163, and often expressed large amounts of IL-17. These data define a terminal ECF pathogenesis in which parasite-driven lymphoproliferation leads to secondary systemic macrophage activation syndrome, mononuclear vasculitis, pulmonary edema, respiratory failure and death. The accompanying macrophage phenotype defined by CD163 and IL-17 is presented in the context of this pathogenesis.

Characterization of leukocyte subsets in buffalo (Bubalus bubalis) with cross-reactive monoclonal antibodies specific for bovine MHC class I and class II molecules and leukocyte differentiation molecules

&NA; Although buffaloes (Bubalus bubalis) are a major component of the livestock industry worldwide, limited progress has been made in the study of the mechanisms regulating the immune response to pathogens and parasites affecting their health and productivity. This has been, in part, attributable to the limited availability of reagents to study immune responses in buffalo. As reported here, a set of cross‐reactive monoclonal antibodies (mAbs), developed against bovine, ovine and caprine leukocyte differentiation molecules (LDM) and major histocompatibility complex (MHC) molecules, were identified and used to compare expression of LDM in Italian and Egyptian buffalo. The results show most of the epitopes identified with the mAbs are conserved on LDM and MHC I and II molecules in both lineages of buffalo. Comparison of the composition of lymphocyte subsets between buffalo and cattle revealed they are similar except for expression of CD2 and CD8 on workshop cluster one (WC1) positive &ggr;&dgr; T cells. In cattle, CD8 is expressed on a subset of CD2+/WC1‐ &ggr;&dgr; T cells that are present in low frequency in blood of young and old animals, whereas, CD8‐/CD2‐/WC1+ &ggr;&dgr; T cells are present in high frequency in young animals, decreasing with age. In the buffalo, CD2 is expressed on a subset of WC1+ &ggr;&dgr; T cells and CD8 is expressed on all WC1+ &ggr;&dgr; T cells. The availability of this extensive set of mAbs provides opportunities to study the immunopathogenesis of pathogens and parasites affecting the health of buffalo. HighlightsMonoclonal antibodies were identified that recognize orthologues of LDMs and MHC I and II molecules in buffalo (Bubalus bubalis).Phenotypic analysis of buffalo lymphocyte subsets showed that all WC1+ &ggr;&dgr; T cells express CD8 with a small subset co‐expressing CD2.The extensive set of cross reactive mAbs will allow for comparison of the similarities and differences in the immune response to pathogens affecting the health of both cattle and buffalo.

Identification of monoclonal antibodies cross-reactive with bottlenose dolphin orthologues of the major histocompatibility complex and leukocyte differentiation molecules

The slow progress in understanding immunotoxic effects of environmental contaminants and their influence on disease susceptibility in whales is largely due to the limited information available on the immune systems and immune function of species included in the Cetancodontamorpha clade. Studies in species in the other major clades included in the Artiodactylamorpha, Ruminantiamorpha, Suinamorpha, and Camelidamorpha have revealed the immune systems are similar, but not identical. The present study was undertaken to expand the available monoclonal antibody reagents needed to gain insight into the composition, function, and evolution of the immune system in Cetancodontamorpha, using the dolphin (Tursiops truncatus) as a model cetacean species. Screening of a set of mAbs that recognize highly conserved epitopes expressed on the major histocompatibility complex (MHC) and leukocyte differentiation molecules (LDMs) in cattle by flow cytometry revealed some of the mAbs recognize epitopes conserved on dolphin orthologues of MHC class I, MHC class II, CD11a, CD14, CD16, CD18, CD163 and CD172a. Comparison of the amino acid sequences of dolphin and bovine orthologues revealed limited changes in sequence have occurred during speciation, suggesting an approach for developing cross-reactive mAbs for use in cetacean research.

Assessment and optimization of Theileria parva sporozoite full-length p67 antigen expression in mammalian cells

Delivery of various forms of recombinant Theileria parva sporozoite antigen (p67) has been shown to elicit antibody responses in cattle capable of providing protection against East Coast fever, the clinical disease caused by T. parva. Previous formulations of full-length and shorter recombinant versions of p67 derived from bacteria, insect, and mammalian cell systems are expressed in non-native and highly unstable forms. The stable expression of full-length recombinant p67 in mammalian cells has never been described and has remained especially elusive. In this study, p67 was expressed in human-derived cells as a full-length, membrane-linked protein and as a secreted form by omission of the putative transmembrane domain. The recombinant protein expressed in this system yielded primarily two products based on Western immunoblot analysis, including one at the expected size of 67 kDa, and one with a higher than expected molecular weight. Through treatment with PNGase F, our data indicate that the larger product of this mammalian cell-expressed recombinant p67 cannot be attributed to glycosylation. By increasing the denaturing conditions, we determined that the larger sized mammalian cell-expressed recombinant p67 product is likely a dimeric aggregate of the protein. Both forms of this recombinant p67 reacted with a monoclonal antibody to the p67 molecule, which reacts with the native sporozoite. Additionally, through this work we developed multiple mammalian cell lines, including both human and bovine-derived cell lines, transduced by a lentiviral vector, that are constitutively able to express a stable, secreted form of p67 for use in immunization, diagnostics, or in vitro assays. The recombinant p67 developed in this system is immunogenic in goats and cattle based on ELISA and flow cytometric analysis. The development of a mammalian cell system that expresses full-length p67 in a stable form as described here is expected to optimize p67-based immunization.

Discovery of a novel species, Theileria haneyi n. sp., infective to equids, highlights exceptional genomic diversity within the genus Theileria : implications for apicomplexan parasite surveillance

A novel apicomplexan parasite was serendipitously discovered in horses at the United States - Mexico border. Phylogenetic analysis based on 18S rDNA showed the erythrocyte-infective parasite to be related to, but distinct from, Theileria spp. in Africa, the most similar taxa being Theileria spp. from waterbuck and mountain zebra. The degree of sequence variability observed at the 18S rDNA locus also suggests the likely existence of additional cryptic species. Among described species, the genome of this novel equid Theileria parasite is most similar to that of Theileria equi, also a pathogen of horses. The estimated divergence time between the new Theileria sp. and T. equi, based on genomic sequence data, is greater than 33 million years. Average protein sequence divergence between them, at 23%, is greater than that of Theileria parva and Theileria annulata proteins, which is 18%. The latter two represent highly virulent Theileria spp. of domestic cattle, as well as of African and Asian wild buffalo, respectively, which differ markedly in pathology, host cell tropism, tick vector and geographical distribution. The extent of genome-wide sequence divergence, as well as significant morphological differences, relative to T. equi justify the classification of Theileria sp. as a new taxon. Despite the overall genomic divergence, the nine member equi merozoite antigen (EMA) superfamily, previously found as a multigene family only in T. equi, is also present in the novel parasite. Practically, significant sequence divergence in antigenic loci resulted in this undescribed Theileria sp. not being detectable using currently available diagnostic tests. Discovery of this novel species infective to equids highlights exceptional diversity within the genus Theileria, a finding with serious implications for apicomplexan parasite surveillance.

Characterization of αβ and γδ T cell subsets expressing IL-17A in ruminants and swine

ABSTRACT As part of our ongoing program to expand immunological reagents available for research in cattle, we developed a monoclonal antibody (mAb) to bovine interleukin‐17A (IL‐17A), a multifunctional cytokine centrally involved in regulating innate and adaptive immune responses. Initial comparative studies demonstrated the mAb recognizes a conserved epitope expressed on orthologues of IL‐17A in sheep, goats and pigs. Comparative flow cytometric analyses of lymphocyte subsets stimulated with phorbol 12‐myristate 13‐acetate (PMA) and ionomycin revealed differences in expression of IL‐17A by CD4, CD8, and &ggr;&dgr; T cells across ruminants and swine species. Results in cattle showed the largest proportion of IL‐17A+ cells were CD4+ followed by &ggr;&dgr; and CD8+ T cells. Further analysis revealed the IL‐17A+ &ggr;&dgr; T cell subset was comprised of WC1.1+, WC1.2+, and WC1‐ subsets. Analysis of the IL‐17A+ CD8+ T cell subset revealed it was comprised of &agr;&bgr; and &ggr;&dgr; T cell subsets. Results in sheep and goats revealed IL‐17A is expressed mainly by CD4+ and CD8+ T cells, with little expression by &ggr;&dgr; T cells. Analysis of IL‐17A+ CD8+ T cells showed the majority were CD8+ &agr;&bgr; in sheep, whereas they were CD8+ &ggr;&dgr; in goats. The majority of the sheep and goat IL‐17A+ &ggr;&dgr; T cells were WC1+. Results obtained in swine showed expression of IL‐17A by CD4, CD8, and &ggr;&dgr; T cell subsets were similar to results reported in other studies. Comparison of expression of IL‐17A with IFN‐&ggr; revealed subsets co‐expressed IL‐17A and IFN‐&ggr; in cattle, sheep, and goats. The new mAb expands opportunities for immunology research in ruminants and swine. HighlightsA bovine specific IL‐17A monoclonal antibody was developed and validated to cross‐react with ovine, caprine and swine species.Flow cytometric analyses of stimulated T cell subsets revealed differences in expression of IL‐17A by CD4, CD8, and &ggr;&dgr; T cells.Comparison of expression of IL‐17A with IFN‐&ggr; revealed T cell subsets co‐expressing IL‐17A and IFN‐&ggr; in cattle, sheep, and goats.Understanding the biology of IL‐17A expression expands knowledge on the cytokine profile of different T cell subsets.

Sporadic juvenile lymphosarcoma in a set of triplet calves: haematopoietic chimerism or chance?

A set of four-month-old Angus-Hereford triplet calves, two freemartin heifers and one steer, was presented for generalised lymphadenomegaly and paraparesis. Cytology was consistent with lymphosarcoma and all calves were serologically negative for bovine leukaemia virus. Postmortem examination and histopathology findings confirmed the diagnosis of sporadic juvenile lymphosarcoma. Due to the rarity of sporadic bovine lymphosarcoma, it is unlikely that these three calves simply, spontaneously, developed lymphosarcoma within a month of one another. A shared genetic predisposition to develop lymphosarcoma is possible, but is unlikely given the synchronous development of disease among the triplets. The most parsimonious explanation is that one calf developed lymphosarcoma in utero and transmitted it to its siblings via placental vascular anastomoses, in the same way haematopoietic chimerism develops in freemartin calves. Regardless of pathogenesis, this case demonstrates that spontaneous bovine leukosis, like enzootic bovine leukosis, occasionally affects multiple animals within a single herd at one time.