Ovine Peripheral Blood Mononuclear Cells Research Articles

Dysregulation of Ovine Toll-Like Receptors 2 and 4 Expression by Hydatid Cyst-Derived Antigens.

Background:Cystic echinococcosis (CE) is a zoonotic disease caused by infection with Echinococcus granulosus. Toll-like receptors (TLRs) as the first line of defense against various parasites play a critical role in sensing and triggering anti-parasite responses.Methods:The study was conducted at the Department of Pathobiology, Faculty of Veterinary Medicine, Ferdowsi University of Mashhad, Iran in 2019. Ovine peripheral blood mononuclear cells (PBMCs) were stimulated with hydatid cyst-derived antigens including hydatid cyst fluid (HCF), germinal layer antigens (GL), somatic and excretory/secretory (ES) products of protoscoleces (PSC). To investigate whether the expression of TLR2 and TLR4 was altered during exposure to these antigens, PBMCs were stimulated with two different concentrations at different time points.Results:After exposure of PBMCs to ES and somatic antigens of protoscoleces (PSC) the expression of TLR2 and TLR4 was down-regulated in comparison with control group. Similarly, HCF markedly down-regulated TLR2 and TLR4 transcripts independent of dose and time. GL antigens significantly down-regulated TLR2, while TLR4 expression was up-regulated as compared with control group.Conclusion:Hydatid cyst-derived antigens could dysregulate the expression of TLR2 and TLR4 in ovine PBMCs, suggesting a possible mechanism to suppress host immunity to establish chronic infection.

Characterization of ovine monocyte activity when cultured with Haemonchus contortus larvae in vitro.

The objective of this study was to identify and characterize cell populations within ovine peripheral blood mononuclear cells (PBMCs) associated with Haemonchus contortus (Hc) larval morbidity and impairment in vitro. Monocytes and lymphocytes were separated from PBMC from parasite-resistant St. Croix (STC) sheep and parasite-susceptible Suffolk (SUF) sheep. Cells were cultured with Hc third stage larvae (L3) for 9h. Larval morbidity was assessed using ATP concentration. Activation status was determined through gene expression analysis and enzyme inhibition. Enzymes arginase-1 (Arg1) and inducible nitric oxide synthase (iNOS) were inhibited using BEC (S-(2-boronoethyl)-I-cysteine) and 1400W (N-(3-(aminomethyl)benzyl)acetamidine), respectively. Larval ATP was lower when cultured with STC-derived monocytes (0.015μmol/L ATP) compared to SUF-derived monocytes (0.067μmol/L ATP) (P<.001), or lymphocytes from either breed (STC: 0.085μmol/L, SUF: 0.112μmol/L ATP) (P<.001). SUF-derived monocytes displayed higher expression of M1 genes, whereas STC-derived monocytes displayed M2 genes continuously. Inhibition of Arg1 decreased monocyte function in both breeds, whereas iNOS inhibition restored SUF-derived monocyte function. Together, these data indicate STC-derived monocytes favour M2 phenotype when exposed to L3, where SUF-derived monocyte function resembled M1 phenotype and described potential for improving Suffolk sheep through modulating inflammatory responses.

Evaluating the effect of TLR4-overexpressing on the transcriptome profile in ovine peripheral blood mononuclear cells

BackgroundToll-like receptor 4 (TLR4) plays an important role in the elimination of Gram-negative bacteria infections and the initiation of antiinflammatory response. Using the technology of pronuclear microinjection, genetically modified (GM) sheep with TLR4 overexpression were generated. Previous studies have shown that these GM sheep exhibited a higher inflammatory response to Gram-negative bacteria infection than wild type (WT) sheep. In order to evaluate the gene expression of GM sheep and study the co-expressed and downstream genes for TLR4, peripheral blood mononuclear cells (PBMC) from TLR4-overexpressing (Tg) and wild type (WT) sheep were selected to discover the transcriptomic differences using RNA-Seq.ResultAn average of 18,754 and 19,530 known genes were identified in the Tg and WT libraries, respectively. A total of 338 known genes and 85 novel transcripts were found to be differentially expressed in the two libraries (p < 0.01). A differentially expressed genes (DEGs) enrichment analysis showed that the GO terms of inflammatory response, cell recognition, etc. were significantly (FDR < 0.05) enriched. Furthermore, the above DEGs were significantly (FDR < 0.05) enriched in the sole KEGG pathway of the Phagosome. Real-time PCR showed the OLR1, TLR4 and CD14 genes to be differentially expressed in the two groups, which validated the DEGs data.ConclusionsThe RNA-Seq results revealed that the overexpressed TLR4 in our experiment strengthened the ovine innate immune response by increasing the phagocytosis in PBMC.

RNA-Sequencing of ovine PBMC after exposure to Haemonchus contortus larval antigen.

Mechanisms of immune activation in effector cells during Haemonchus contortus infection in sheep are currently unknown. Microarray experiments have been performed on tissues of Hcontortus infected sheep of varying parasite resistance during early and late points of infection, but not in immune effector cells. The purpose of this study was to compare early gene activation in peripheral blood mononuclear cells (PBMC) from primed parasite susceptible (Suffolk) and resistant (St. Croix) sheep in response to Hcontortus larval antigen (HcLA). Peripheral blood mononuclear cells were cultured for 6hours with HcLA, and RNA-sequencing was performed. St. Croix PBMC upregulated 499 unique genes in response to HcLA while Suffolk PBMC upregulated 130 unique genes and 25 genes were shared between the two breeds. St. Croix PBMC had increased expression of genes associated with immune function, signal transduction, response to stress and others. In addition, while mechanisms of innate recognition of Hcontortus are unknown, multiple pattern recognition receptors were found to be upregulated in St. Croix PBMC cultured with HcLA and none were found to be upregulated in Suffolk PBMC. These patterns of immune gene activation may contribute to St. Croix's rapid response and ability to resist Hcontortus infection.

Fasciola hepatica Infection in Cattle: Analyzing Responses of Peripheral Blood Mononuclear Cells (PBMC) Using a Transcriptomics Approach.

The parasitic helminth Fasciola hepatica (liver fluke) causes economic loss to the livestock industry globally and also causes zoonotic disease. New control strategies such as vaccines are urgently needed, due to the rise of drug resistance in parasite populations. Vaccine development requires a comprehensive understanding of the immunological events during infection. Previous in vivo studies by our group have investigated global differentially expressed genes (DEGs) in ovine peripheral blood mononuclear cells (PBMC) in response to both acute and chronic F. hepatica infection. This work demonstrated that pathways involved in the pathogenesis of ovine fasciolosis included fibrosis, inhibition of macrophage nitric oxide production, and antibody isotype switching, among others. Transcriptomic changes in PBMC populations following F. hepatica infection in cattle, in which the disease phenotype is quite different, have not yet been examined. Using RNA sequencing we investigated gene expression changes in PBMC isolated from 9 non-infected and 11 F. hepatica-experimentally-infected calves immediately before infection, at 1 and at 14 weeks post-infection. Longitudinal time-course comparisons between groups revealed 21 and 1,624 DEGs driven exclusively by F. hepatica infection in cattle at acute and chronic stages, respectively. These results show that fewer DEGs at the acute stage of infection can be identified in cattle, as compared with sheep. In addition, the log2 fold-changes of these DEGs were relatively low (−1 to 3) reflecting the different clinical presentation of F. hepatica infection in cattle. Gene pathways for hepatic fibrosis and hepatic cholestasis along with apoptosis of antigen-presenting cells were enriched at chronic stages. Our results reflect the major differences in the disease phenotype between cattle and sheep and may indicate pathways to target in vaccine development.

Network Analysis of the Systemic Response to Fasciola hepatica Infection in Sheep Reveals Changes in Fibrosis, Apoptosis, Toll-Like Receptors 3/4, and B Cell Function.

The Trematode Fasciola hepatica is an important cause of disease in livestock and in man. Modulation of immunity is a critical strategy used by this parasite to facilitate its long-term survival in the host. Understanding the underlying mechanisms at a system level is important for the development of novel control strategies, such as vaccination, as well as for increasing general understanding of helminth-mediated immunoregulation and its consequences. Our previous RNA sequencing work identified a large number of differentially expressed genes (DEG) from ovine peripheral blood mononuclear cells (PBMCs) at acute and chronic stages of F. hepatica infection, and yielded important information on host–parasite interaction, with particular reference to the immune response. To extend our understanding of the immunoregulatory effects of this parasite, we employed InnateDB to further analyze the DEG dataset and identified 2,458 and 224 molecular interactions in the context of innate immunity from the acute and chronic stages of infection, respectively. Notably, 458 interactions at the acute stage of infection were manually curated from studies involving PBMC-related cell-types, which guaranteed confident hypothesis generation. NetworkAnalyst was subsequently used to construct and visualize molecular networks. Two complementary strategies (function-first and connection-first) were conducted to interpret the networks. The function-first approach highlighted subnetworks implicated in regulation of Toll-like receptor 3/4 signaling in both acute and chronic infections. The connection-first approach highlighted regulation of intrinsic apoptosis and B-cell receptor-signaling during acute and chronic infections, respectively. To the best of our knowledge, this study is the first system level analysis of the regulation of host innate immunity during F. hepatica infection. It provides insights into the profound changes induced by F. hepatica infection that not only favors parasite survival into chronic infection but also impedes the host’s immune response to other pathogens, and render vaccination against fasciolosis a difficult challenge. The information provided will be useful in the design of specific vaccine protocols to overcome parasite-mediated immunoregulation and in furthering general understanding of the interplay between helminth infection and host immune systems.

Transcriptomic Study on Ovine Immune Responses to Fasciola hepatica Infection.

BackgroundFasciola hepatica is not only responsible for major economic losses in livestock farming, but is also a major food-borne zoonotic agent, with 180 million people being at risk of infection worldwide. This parasite is sophisticated in manipulating the hosts’ immune system to benefit its own survival. A better understanding of the mechanisms underpinning this immunomodulation is crucial for the development of control strategies such as vaccines.Methodology/principal findingsThis in vivo study investigated the global gene expression changes of ovine peripheral blood mononuclear cells (PBMC) response to both acute & chronic infection of F. hepatica, and revealed 6490 and 2364 differential expressed genes (DEGS), respectively. Several transcriptional regulators were predicted to be significantly inhibited (e.g. IL12 and IL18) or activated (e.g. miR155-5p) in PBMC during infection. Ingenuity Pathway Analysis highlighted a series of immune-associated pathways involved in the response to infection, including ‘Transforming Growth Factor Beta (TGFβ) signaling’, ‘Production of Nitric Oxide in Macrophages’, ‘Toll-like Receptor (TLRs) Signaling’, ‘Death Receptor Signaling’ and ‘IL17 Signaling’. We hypothesize that activation of pathways relevant to fibrosis in ovine chronic infection, may differ from those seen in cattle. Potential mechanisms behind immunomodulation in F. hepatica infection are a discussed.SignificanceIn conclusion, the present study performed global transcriptomic analysis of ovine PBMC, the primary innate/adaptive immune cells, in response to infection with F. hepatica, using deep-sequencing (RNAseq). This dataset provides novel information pertinent to understanding of the pathological processes in fasciolosis, as well as a base from which to further refine development of vaccines.

Assessment of reference genes for reliable analysis of gene transcription by RT-qPCR in ovine leukocytes

With the availability of genetic sequencing data, quantitative reverse transcription PCR (RT-qPCR) is increasingly being used for the quantification of gene transcription across species. Too often there is little regard to the selection of reference genes and the impact that a poor choice has on data interpretation. Indeed, RT-qPCR provides a snapshot of relative gene transcription at a given time-point, and hence is highly dependent on the stability of the transcription of the reference gene(s). Using ovine efferent lymph cells and peripheral blood mono-nuclear cells (PBMCs), the two most frequently used leukocytes in immunological studies, we have compared the stability of transcription of the most commonly used ovine reference genes: YWHAZ, RPL-13A, PGK1, B2M, GAPDH, HPRT, SDHA and ACTB. Using established algorithms for reference gene normalization “geNorm” and “Norm Finder”, PGK1, GAPDH and YWHAZ were deemed the most stably transcribed genes for efferent leukocytes and PGK1, YWHAZ and SDHA were optimal in PBMCs. These genes should therefore be considered for accurate and reproducible RT-qPCR data analysis of gene transcription in sheep.

Suppression of ovine lymphocyte activation by Teladorsagia circumcincta larval excretory-secretory products

Teladorsagia circumcincta is an important pathogenic nematode of sheep. It has been demonstrated previously that stimulation of murine T lymphocytes with excretory-secretory (ES) products derived from fourth stage larvae of T. circumcincta (Tci-L4-ES) results in de novo expression of Foxp3, a transcription factor intimately involved in regulatory T cell function. In the current study, Foxp3+ T cell responses in the abomasum and the effects of Tci-L4-ES on ovine peripheral blood mononuclear cells (PBMC) following T. circumcincta infection were investigated. T. circumcincta infection resulted in a significant increase in numbers of abomasal Foxp3+ T cells, but not an increase in the proportion of T cells expressing Foxp3. Unlike in mice, Tci-L4-ES was incapable of inducing T cell Foxp3 expression but instead suppressed mitogen-induced and antigen-specific activation and proliferation of ovine PBMC in vitro. This effect was heat labile, suggesting that it is mediated by protein(s). Suppression was associated with up-regulation of interleukin-10 (IL-10) mRNA, and specific monoclonal antibody neutralisation of IL-10 resulted in a 50% reduction in suppression, indicating involvement of the IL-10 signaling pathway. Suppression was significantly reduced in PBMC isolated from T. circumcincta infected vs. helminth-naïve lambs, and this reduction in suppression was associated with an increase in Tci-L4-ES antigen-specific T cells within the PBMC. In conclusion, we have identified a mechanism by which T. circumcincta may modulate the host adaptive immune response, potentially assisting survival of the parasite within the host. However, the impact of Tci-L4-ES-mediated lymphocyte suppression during T. circumcincta infection remains to be determined.

The measurement of three cytokine transcripts in naïve and sensitized ovine peripheral blood mononuclear cells following in vitro stimulation with bluetongue virus serotype-23

Bluetongue (BT), a serious disease of sheep and some wild ruminants, is caused by bluetongue virus (BTV), a member of the family, Reoviridae. The current research thrust for controlling BT is on development of efficient vaccines, necessitating clear understanding of ovine immunology. At present, comparative studies on cytokine gene expression profiles of naïve and BTV-sensitized peripheral blood mononuclear cells (PBMC) in sheep have not been clearly understood. In the present study, PBMC from naïve and BEI-inactivated-saponin-adjuvanted BTV-1 vaccinated sheep were stimulated in vitro with heterologous BTV-23. At various intervals, RT-qPCR was carried out to estimate cytokine (interferon-gamma, interleukin-12 and interleukin-2) mRNA expressions that are linked to cell-mediated immunity. The results showed that PBMC cytokine profiles were relatively increased both temporally and quantitatively in immunized sheep PBMC compared to naïve ones, suggesting that BTV-1 vaccination may prime immune system that can cross-react with BTV-23 antigens.

Relative quantitative kinetics of interferon-gamma and interleukin-10 mRNA and protein production by activated ovine peripheral blood mononuclear cells

Interferon-gamma (IFN-γ) and interleukin (IL)-10 are cross-regulatory cytokines capable of driving and controlling the adaptive host immune response. The inter-relationship between IFN-γ and IL-10 expression has not been defined in sheep despite biological evidence suggesting that they perform similar functions to their orthologues described in other species. To address this, we have developed a quantitative (q)PCR method to assess relative levels of IFN-γ and IL-10 mRNA expression in activated ovine peripheral blood mononuclear cells (PBMC) and compared the kinetics of mRNA expression with amounts of cytokine secreted by the cells over a 96 h period. PBMC were collected from sheep immunised with the nominal antigen ovalbumin (Ova) and re-stimulated in vitro with antigen and the T cell mitogen concanavalin A (ConA). The recall response to antigen was characterised by a single peak in IFN-γ mRNA expression at 48 h of culture (13-fold increase over unstimulated cells) and relatively lower expression of IL-10 mRNA (average 2–3-fold increase over the 96 h culture period). Antigen-driven IFN-γ protein concentration was greatest at the end of the culture period (96 h) whereas IL-10 protein level was not elevated above that observed in unstimulated cells. The typical response to ConA was greater for both cytokines, with IFN-γ mRNA expression peaking at 6 h of culture (133-fold increase) then declining rapidly whereas IL-10 mRNA expression peaked at 24 h (16-fold increase) and declined more gradually. Despite these differences in the relative kinetics of mRNA expression in mitogen-activated PBMC, the typical pattern of protein expression of the two cytokines was similar. Both showed a gradual rise in protein concentration starting from 12 h of culture which was still rising at the end of the culture period (96 h). These data demonstrate that the kinetics of mRNA expression for IFN-γ and IL-10 in activated ovine PBMC do not necessarily correlate with detectable protein in culture.

Global suppression of mitogen-activated ovine peripheral blood mononuclear cells by surface protein activity from Mycoplasma ovipneumoniae

Mycoplasma ovipneumoniae is associated with chronic non-progressive pneumonia of sheep and goats. As with many other mycoplasmas involved in animal diseases, protective immune responses have not been achieved with vaccines, even though antibody responses can be obtained. This study focuses on characterizing the interaction of M. ovipneumoniae with ovine PBMC using carboxy-fluorescein-succinimidyl-ester (CFSE) loading and flow cytometry to measure lymphoid cell division. M. ovipneumoniae induced a strong in vitro polyclonal suppression of CD4 +, CD8 +, and B blood lymphocyte subsets. The suppressive activity could be destroyed by heating to 60 °C, and partially impaired by formalin and binary ethyleneimine treatment that abolished its viability. The activity resided on the surface-exposed membrane protein fraction of the mycoplasma, since mild trypsin treatment not affecting viability was shown to reduce suppressive activity. Trypsin-treated mycoplasma regained suppressive activity once the mycoplasma was allowed to re-synthesize its surface proteins. Implications for the design of vaccines against M. ovipneumoniae are discussed.

Ovine CD16+/CD14-blood lymphocytes present all the major characteristics of natural killer cells

Natural killer (NK) cells have a key role in the innate immune response against pathogens because of their cytotoxic properties and production of interferon-gamma (IFN-γ). Some insight into ruminant NK cell biology has been gained through the characterization of bovine NK cells as NKp46+/CD3− cells. However, ovine NK cells have been little studied because of the lack of specific antibodies. Most NK cells in humans and cattle express CD16. We found that an antibody against human CD16 that cross-reacts with bovine NK cells also recognizes cell populations in ovine peripheral blood mononuclear cells. Using double labelling with CD14 revealed the same profile as described in other species, and we identified a putative NK cell population. We therefore sorted this ovine CD16+/CD14− cell population and tested it for NK cell characteristics. More than 80% of sorted CD16+/CD14− cells expressed perforin. After a week of culture in the presence of IL-2 and IL-15, ovine CD16+/CD14− cells had become large cells with intra-cytoplasmic granules containing perforin, and the vast majority displayed an activated CD2−/low/CD25+/CD8+ phenotype, as observed for bovine NKp46+/CD3− cells. Moreover, these cells expressed transcripts for the NKp46 receptor, and were cytotoxic in a CD16-mediated redirected lysis assay against a murine cell line, P815, and in a direct lysis assay against the ovine cell line, IDO5. Finally, ovine CD16+/CD14− cells having expanded for 7 days in culture secreted IFN-γ in response to IL-12 in a dose-dependent manner. Taken together, these findings led us to conclude that the ovine CD16+/CD14− lymphocyte sub-population displays the phenotype and functional characteristics of NK cells.

Safety and immunogenicity of myxoma virus as a new viral vector for small ruminants

Myxoma virus (MYXV), a leporide-specific poxvirus, represents an attractive candidate for the generation of safe and non-replicative vaccine vectors for other species. With the aim of developing new recombinant vaccines for ruminants, we evaluated the safety and the immunogenicity of recombinant MYXV in sheep. In vitro studies indicated that ovine primary fibroblasts were not permissive for MYXV and that infection of ovine peripheral blood mononuclear cells occurred at a low rate. Although non-specific activation significantly improved the susceptibility of lymphocytes, MYXV infection remained abortive. Histological and immunohistochemical examination at the inoculation sites revealed the development of an inflammatory process and allowed the detection of sparse infected cells in the dermis. In addition, inoculated sheep developed an antibody response directed against MYXV and the product of the transgene. Overall, these results provide the first line of evidence on the potential of MYXV as a viral vector for ruminants.

Ovine plasma prion protein levels show genotypic variation detected by C-terminal epitopes not exposed in cell-surface PrPC

Ovine PBMCs (peripheral blood mononuclear cells) express PrP(C) [cellular PrP (prion-related protein)] and have the potential to harbour and release disease-associated forms of PrP during scrapie in sheep. Cell-surface PrP(C) expression by PBMCs, together with plasma PrP(C) levels, may contribute to the regulatory mechanisms that determine susceptibility and resistance to natural scrapie in sheep. Here, we have correlated cell-surface PrP(C) expression on normal ovine PBMCs by FACS with the presence of PrP(C) in plasma measured by capture-detector immunoassay. FACS showed similar levels of cell-surface PrP(C) on homozygous ARR (Ala136-Arg154-Arg171), ARQ (Ala136-Arg154-Gln171) and VRQ (Val136-Arg154-Gln171) PBMCs. Cell-surface ovine PrP(C) showed modulation of N-terminal epitopes, which was more evident on homozygous ARR cells. Ovine plasma PrP(C) levels showed genotypic variation and the protein displayed C-terminal epitopes not available in cell-surface PrP(C). Homozygous VRQ sheep showed the highest plasma PrP(C) level and homozygous ARR animals the lowest. For comparison, similar analyses were performed on normal bovine PBMCs and plasma. PrP(C) levels in bovine plasma were approx. 4-fold higher than ovine homozygous ARQ plasma despite similar levels of PBMC cell-surface PrP(C) expression. Immunoassays using C-terminal-specific anti-PrP monoclonal antibodies as capture and detector reagents revealed the highest level of PrP(C) in both ovine and bovine plasma, whilst lower levels were detected using N-terminal-specific monoclonal antibody FH11 as the capture reagent. This suggested that a proportion of plasma PrP(C) was N-terminally truncated. Our results indicate that the increased susceptibility to natural scrapie displayed by homozygous VRQ sheep correlates with a higher level of plasma PrP(C).

Molecular cloning and mRNA tissue-expression of two isoforms of the ovine costimulatory molecule CD80 (B7-1)

Using RT-PCR and rapid amplification of cDNA ends (RACE), we cloned two putative alternatively spliced transcripts of the sheep CD80 (B7-1) molecule that encode both transmembrane (TM) and secreted (s) forms of CD80 protein. Comparison of the amino acid sequence of the TM form of ovine CD80 with the sequence of cattle, swine and human CD80 indicated that the deduced protein had a higher degree of similarity to cattle (87% of amino acid identity) than to pig (68%) and human sequence (53% of homology). In tissues, RT-PCR using primers for the TM and the sCD80 transcripts indicated that the expression of both CD80 transcripts was almost exclusively expressed in the hematolymphoid system, with the exception of the uterus. The sCD80 transcript was expressed in peripheral blood mononuclear cells (PBMC), uterus and lymph node, whereas the TM-CD80 transcript was very weakly detected only in PBMC cells. Our result indicates that mRNA transcripts encoding both membrane-bound and secreted CD80 proteins are expressed in sheep like in other animals. However, in contrast with the CD80 molecules from other species, the secreted form of sheep CD80 seems to be the predominant form expressed in the ovine PBMC and other tissues, suggesting that the TM-CD80 represents a rare transcript in this species. Interestingly, the expression of both forms of the CD80 molecule was not affected by treatment of sheep PBMC with Concanavalin A (ConA), as detected by RT-PCR. This is the first report describing the identification of a B7 costimulatory transcript in sheep.

Conformational variation between allelic variants of cell-surface ovine prion protein.

The distribution of prion infectivity and PrPSc between peripheral lymphoid tissues suggests their possible haematogenic spread during the progression of natural scrapie in susceptible sheep. Since ovine PBMCs (peripheral blood mononuclear cells) express PrPC, they have the potential to carry or harbour disease-associated forms of PrP. To detect the possible presence of disease-associated PrP on the surface of blood cells, an understanding is required of the conformations that normal ovine cell-surface PrPC may adopt. In the present study, we have used monoclonal antibodies that recognize epitopes in either the N- or C-terminal portions of PrP to probe the conformations of PrPC on ovine PBMCs by flow cytometry. Although PBMCs from scrapie-susceptible and -resistant genotypes of sheep expressed similar levels of cell-surface PrPC, as judged by their reactivity with N-terminal-specific anti-PrP monoclonal antibodies, there was considerable genotypic heterogeneity in the region between helix-1 and residue 171. Cells from PrP-VRQ (V136R154Q171) sheep showed uniform reactivity with monoclonal antibodies that bound to epitopes around helix-1, whereas cells from PrP-ARQ (A136R154Q171) and PrP-ARR (A136R154R171) sheep showed variable binding. The region between b-strand-2 and residue 171, which includes a YYR motif, was buried or obscured in cell-surface PrPC on PBMCs from scrapie-susceptible and -resistant sheep. However, an epitope of PrPC that is influenced by residue 171 was more exposed on PBMCs from PrP-VRQ sheep than on PBMCs from the PrP-ARQ genotype. Our results highlight conformational variation between scrapie-susceptible and -resistant forms of cell-surface PrPC and also between allelic variants of susceptible genotypes.

Anaplasma phagocytophilum reduces neutrophil apoptosis in vivo.

Ovine neutrophils spontaneously underwent apoptosis during culture in vitro, as assessed by morphological changes and exposure of annexin V binding sites on their cell surfaces. The addition of conditioned medium from concanavalin A-treated ovine peripheral blood mononuclear cells (PBMC) could partially protect against this progression into apoptosis, but dexamethasone and sodium butyrate could not. Actinomycin D accelerated the rate at which ovine neutrophils underwent apoptosis. Neutrophils isolated from sheep experimentally infected with Anaplasma phagocytophilum showed significantly delayed apoptosis during culture ex vivo, and the addition of conditioned medium from PBMC to these cells could not delay apoptosis above the protective effects observed after in vivo infection. The ability of neutrophils from A. phagocytophilum-infected sheep to activate a respiratory burst was increased compared to the activity measured in neutrophils from uninfected sheep, but chemotaxis was decreased in neutrophils from infected sheep. These data are the first demonstration that in vivo infection with A. phagocytophilum results in changes in rates of apoptosis of infected immune cells. This may help explain how these bacteria replicate in these normally short-lived cells.

Effects of endotoxin exposure on cationic amino acid transporter function in ovine peripheral blood mononuclear cells.

Rodent models of sepsis differ from clinical human disease in that humans make substantially less whole-body nitric oxide and have different cellular responses to endotoxin. Sheep, when exposed to endotoxin, behave in a manner more similar to humans. Many studies of rodent peripheral blood mononuclear cells (PBMCs) exposed to endotoxin demonstrate increased cationic amino acid transporter function (particularly through the y+ transporter) to supply arginine substrate to upregulated nitric oxide synthase. Whether this is true in sheep is not known. We have studied cationic amino acid transport in sheep PBMCs stimulated with endotoxin, using labelled lysine. PBMCs stimulated both in vitro and in vivo show an initial reduction in total and y+ lysine transport (after 1-2 h exposure to endotoxin): a previously undescribed effect of endotoxin. In in vitro activated cells, the reduction in y+ transport was prevented by the lipoxygenase inhibitor, nordihydroguaretic acid (NDGA), and the phospholipase inhibitor 4-bromophenacyl bromide (4-BPAB), but not cyclohexamide or a number of other inhibitors of intracellular second-messenger pathways. In contrast after 14 h incubation, the expected increase in total and y+ lysine transport was seen. The increase in y+ transport could be prevented by cyclohexamide, dexamethasone, ibuprofen, the protein kinase C inhibitor sphingosine, NDGA and 4-BPAB. These results suggest that in response to endotoxin exposure there is an initial decrease in y+ activity mediated by a lipoxygenase product, followed by a substantial increase in y+ activity mediated by the products of either cyclo-oxygenase or lipoxygenase. Cyclo-oxygenase and/or lipoxygenase inhibition might be useful in reducing arginine transport, and hence nitric oxide production, in these cells.

Production of Tumour Necrosis Factor-alpha (TNF-α) and Reactive Nitrogen Intermediates by Ovine Peripheral Blood Leucocytes Stimulated by Ehrlichia (Cytoecetes) phagocytophila

Ehrlichia (Cytoecetes) phagocytophila, the causative agent of tick-borne fever in sheep and pasture fever in cattle, is an immunosuppressive, obligately intracellular rickettsia that invades granulocytes and monocytes of ruminants. Infected animals are known to suffer from a number of secondary infections. The mechanisms of immunosuppression are believed to be associated with physical or functional damage to leucocytes and the release of immunosuppressive substances. In the present study, the effects of E. phagocytophila on the production of tumour necrosis factor-alpha (TNF-α) and reactive nitrogen intermediates by ovine peripheral blood mononuclear cells (PBMCs) were investigatedin vivo and in vitro. The concentration of TNF-α and nitrate in ovine sera were significantly increased during infection with E. phagocytophila, peak concentrations occurring at the peak period of rickettsiaemia. The addition of E. phagocytophila to cell cultures enhanced in-vitro production of TNF-α and nitric oxide by normal ovine PBMCs.