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peer-reviewed Bovine mastitis is an inflammation-driven disease of the bovine mammary gland that costs the global dairy industry several billion dollars per annum. Because disease susceptibility is a multi-factorial complex phenotype, an integrative biology approach is required to dissect the molecular networks involved. Here, we report such an approach, using next generation sequencing combined with advanced network and pathway biology methods to simultaneously profile mRNA and miRNA expression at multiple time-points (0, 12, 24, 36 and 48h) in both milk and blood FACS-isolated CD14+ monocytes from animals infected in vivo with Streptococcus uberis. More than 3,700 differentially expressed (DE) genes were identified in milk-isolated monocytes (MIMs), a key immune cell recruited to the site of infection during mastitis. Up-regulated genes were significantly enriched for inflammatory pathways, while down-regulated genes were enriched for non-glycolytic metabolic pathways. Monocyte transcriptional changes in the blood, however, were more subtle but highlighted the impact of this infection systemically. Genes up-regulated in blood-isolated-monocytes (BIMs) showed a significant association with interferon and chemokine signalling. Furthermore, twenty-six miRNAs were differentially expressed in MIMs and three in BIMs. Pathway analysis revealed that predicted targets of down-regulated miRNAs were highly enriched for roles in innate immunity (FDR < 3.4E-8) in particular TLR signalling, while up-regulated miRNAs preferentially targeted genes involved in metabolism. We conclude that during S. uberis infection miRNAs are key amplifiers of monocyte inflammatory response networks and repressors of several metabolic pathways. This study was funded in part by Teagasc RMIS 6018 and United States Department of Agriculture ARS funding 3625-32000-102-00. NL is supported by a Teagasc Walsh Fellowship.

Paratuberculosis is a chronic intestinal disease of ruminant animals caused by Mycobacterium avium subsp. paratuberculosis (MAP). A hallmark of paratuberculosis is a transition from a cell-mediated Th1 type response to a humoral Th2 response with the progression of disease from a subclinical to clinical state. The objective of this study was to investigate the expression of two crucial molecules in T cell function, ZAP-70 (zeta-chain-associated protein of 70 kDa) and CTLA-4 (cytotoxic T-lymphocyte antigen-4), in cows naturally infected with MAP. Peripheral blood mononuclear cells (PBMCs) isolated from control non-infected cows (n=5), and cows in subclinical (n=6) and clinical stages of paratuberculosis (n=6) were cultured alone (medium only), and with concanavalin A, and a whole cell sonicate of MAP for 24, 72 and 144 h to measure the dynamic changes of ZAP-70 and CTLA-4 expression on CD4, CD8, and gamma delta (γδ) T cells. Flow cytometry was also performed to measure ZAP-70 phosphorylation to examine proximal T cell receptor signaling in animals of different disease status. The surface expression of CTLA-4 was increased in animals in subclinical stage of infection while levels of ZAP-70 were decreased in CD4+ T cells of both subclinical and clinical animals, indicating a change in T cell phenotype with disease state. Interestingly, proximal T cell receptor signaling was not altered in infected animals. This study demonstrated changes in crucial signaling molecules in animals infected with MAP, thereby elucidating T cell alterations associated with disease progression.

Stimulation of lymphocyte proliferation using mitogens or specific antigens is a method that is used frequently to assess immune responsiveness. While useful, lymphocyte blastogenesis, or [ 3 H ] -thymidine incorporation, provides little information regarding the response of specific subsets to the stimulant. Here, we report that the fluorescent cell membrane probe, PKH2, is a useful tool for measuring the proliferation of porcine lymphocyte subpopulations by utilizing multicolor flow cytometry. For this study, mitogen-induced proliferation of porcine peripheral blood mononuclear cells (PBMCs) was measured using [ 3 H ] -thymidine incorporation as well as a flow cytometric-based proliferation assay. From the [ 3 H ] -thymidine incorporation data alone, it was observed that PBMC stimulated with either concanavalin A (Con A), phytohemagglutinin (PHA) or pokeweed mitogen (PWM) demonstrated greater proliferation on day 3 than on day 5 of culture. Using the PKH dye and flow cytometric analysis, the responsiveness of specific lymphocyte subsets to mitogen stimulation was detected. The predominant subsets of porcine lymphocytes responding to Con A or PHA stimulation were CD4 + CD8 + , CD4 − CD8α hi , CD4 − CD8α lo and γδ TCR + cells. PWM stimulation induced responses by CD4 + CD8 + , CD4CD8α hi but not by CD4 − CD8α lo or γδ TCR + cells. Con A stimulation resulted in a sustained proliferation of CD8α hi cells over the 5-day period while PHA stimulation resulted in proliferation that peaked within the first 3 days. Little or no proliferative responses were detected within the IgM + population (e.g. B cells). This is the first study to define the contribution of individual lymphocyte subsets to mitogen-induced proliferation of porcine PBMCs.

The objective of this study was to develop a rapid and reliable method for flow cytometric analysis of porcine whole blood cells. Fifty-microliters of heparin- or EDTA-treated whole blood was added to wells of a round-bottom 96-well microtitration plate. Each well contained 10 microl of an appropriate dilution of four different antibodies (40 microl total; two primary monoclonal antibodies and two fluorescent-labeled secondary antibodies). For convenience, the antibody mixture could be added to plates 1-2 days prior to assay and stored at 4 degrees C. Once whole blood was added to wells, plates were mixed gently, placed in a sealed bag and incubated in the dark at room temperature for 20 min. Contents of wells were then transferred to polystyrene tubes containing 2 ml of 1.5% formalin in distilled water and mixed gently. Cells were fixed for a minimum of 30 min and then stored in the dark at 4 degrees C until analysis by flow cytometry. Analysis of cell samples may be done up to 3 days after fixation. Results indicate that the percentages of Class I, Class II, CD3, CD8, CD4, CD45, monocyte, gamma-delta T-cell populations, and total number of granulocytes identified using this method were comparable to standard values or to values obtained following separation of white blood cells from red blood cells. The percentage of labeled B-cells was lower than standard values. Total assay time from receipt of blood to acquisition of data by flow cytometry required less than 2 h. This modified assay was shown to be simple, reliable, and useful for screening large numbers of porcine samples in a minimal period of time.

Brachyspira (Serpulina) hyodysenteriae infection of pigs (swine dysentery) causes a mucohemorrhagic diarrhea resulting in significant economic losses for producers. A commercial vaccine consisting of a proteinase-digested bacterin has shown efficacy in the reduction of disease due to B. hyodysenteriae. Vaccines consisting of whole cell bacterins, however, generally fail to protect pigs from disease. In the present study, cellular immune responses induced by a proteinase-digested bacterin were compared to responses induced by a whole cell sonicate antigen preparation. In addition, usage of either squalene or Freund's incomplete adjuvants in combination with each antigen preparation was also compared. Both antigen preparations induced significant cellular immune responses as measured by in vitro (IFN-gamma production and T cell proliferation) and in vivo methods (DTH responses). No significant differences were detected in proliferative, interferon-gamma (IFN-gamma), or delayed type hypersensitivity (DTH) responses by pigs receiving either adjuvant or antigen preparation. T cells (CD3(+)) but not B cells from vaccinated animals proliferated in response to in vitro stimulation with B. hyodysenteriae antigen. CD8(+) (single positive and CD4/CD8 double positive) and gammadelta(+) T cells were particularly responsive. In addition, high percentages of both CD8 single positive and CD4/CD8 double positive cells were detected in antigen-stimulated cultures. These findings demonstrate the unique sensitivity of porcine CD8(+) T cells to priming for recall response by vaccination with a proteinase-digested B. hyodysenteriae bacterin.

Twelve dairy cows infected with Mycobacterium avium subsp. paratuberculosis were monitored for lymphocyte subsets and expression of adhesion molecules on cells in blood and milk at parturition and at intervals up to 21 days post-partum. Using fluorescent antibody labeling of cells and analysis by flow cytometry, we determined percentages of T cell subsets (CD4+, CD8+, gammadelta+) and expression of adhesion molecules (CD62L, LFA-1, LPAM-1, and CD44) on cells from blood and milk of these cows. Significantly higher percentages of CD8+ cells were found in milk than in blood at all time points; there were no significant differences in percentages of CD4+ or gammadelta+ cells. CD62L, LFA-1, and LPAM-1 were expressed on a significantly higher percentage of all T cell subsets in milk than in blood at various times after parturition. No differences were seen in expression of CD44. Increased percentages of T lymphocytes expressing adhesion molecules in milk compared to blood suggest that a migratory population of cells is being selectively recruited to the mammary gland from the circulation.

Effects of increased dietary energy and protein on the composition and functional capacities of blood mononuclear leukocyte populations from milk replacer-fed calves were investigated. Holstein bull calves (average age: 4.2 d; n = 19) were assigned randomly to one of two treatment groups. Treatment 1 calves (n = 9) were fed a 20% crude protein, 20% fat milk replacer at a rate of 1.4% body weight of dry matter/d for 8 wk, whereas treatment 2 calves (n = 10) were fed a 30% crude protein, 20% fat milk replacer at a rate of 2.5% body weight of dry matter per day. Composition and functional capacities of mononuclear leukocyte populations from blood samples collected at 4, 18, 32, 46, and 60 d of age were characterized by flow cytometry and ex vivo cell function assays. From 11 to 60 d of age, the mean daily weight gain of treatment 2 calves (1.20 kg/d) was greater than daily weight gain of treatment 1 calves (0.55 kg/d). At 60 d of age, the mean body weight of treatment two calves was 53% (39 kg) greater than the mean body weight of treatment 1 calves. Total numbers of blood leukocytes and the composition of the mononuclear leukocyte population were unaffected by the plane of nutrition. Mitogen-induced DNA-synthesis and immunoglobulin M secretion also were unaffected by dietary treatment. Blood mononuclear leukocytes from calves on intensified diets, however, produced less interferon-gamma and more inducible nitric oxide, suggesting that increased dietary energy and protein affects specific aspects of leukocyte function associated with cell-mediated immunity. The impact of altered interferon-gamma and NO production on the calf s susceptibility to infectious disease are not known. Mononuclear leukocyte populations from all calves also demonstrated age-related changes in composition and functional capacity, likely reflecting natural exposure to infectious agents and maturation of the calfs immune system.

Despite highly successful eradication efforts in several countries, Mycobacterium bovis infection of cattle remains a significant health concern worldwide. Immune mechanisms of resistance to and/or clearance of M. bovis infection of cattle, however, are unclear. Recent studies have provided evidence supporting a role for CD4(+), CD8(+), and gammadelta TCR(+) T cells in the response of cattle to M. bovis. In the present study, we utilized a flow cytometric-based proliferation assay to determine the relative contribution of individual lymphocyte subsets in the response to M. bovis infection and/or sensitization with mycobacterial purified protein derivative (PPD). Peripheral blood mononuclear cells (PBMC) from M. bovis-infected cattle proliferated in response to in vitro stimulation with M. bovis PPD. CD4(+) T cells and gammadelta TCR(+) cells were the predominate subsets of lymphocytes responding to PPD. gammadelta TCR(+) cells also proliferated in non-stimulated cultures; however, the gammadelta TCR(+) cell proliferative response of infected cattle was significantly (p

White-tailed deer are significant wildlife reservoirs of Mycobacterium bovis for cattle, predators, and, potentially, humans. Infection of cattle with M. bovis stimulates an antigen-specific T-cell response, with both CD4(+) and CD8(+) cells implicated in protective immunity. Few studies, however, have examined lymphocyte subset responses to experimental M. bovis infection of white-tailed deer. In this study, a flow cytometric proliferation assay was used to determine the relative contribution of individual peripheral blood mononuclear cell subsets of M. bovis-infected white-tailed deer in the recall response to M. bovis antigen. Naive deer were challenged with M. bovis by cohabitation with infected deer. These M. bovis-challenged deer developed significant in vivo (delayed-type hypersensitivity) and in vitro (proliferative) responses to M. bovis purified protein derivative (PPD). At necropsy, typical tuberculous lesions containing M. bovis were detected within lungs and lung-associated lymph nodes of infected deer. The predominant subset of lymphocytes that proliferated in response to in vitro stimulation with PPD was the CD4(+) subset. Minimal proliferative responses were detected from CD8(+), gamma delta TCR(+), and B-cells. Addition of monoclonal antibodies specific for MHC II antigens, but not MHC I or CD1 antigens, abrogated the proliferative response. Together, these findings indicate that while CD4(+) cells from infected deer proliferate in the recall response to M. bovis antigens, this response is not sufficient to clear M. bovis and immunologic intervention may require stimulation of alternate subsets of lymphocytes.

Mycobacterium avium subsp. paratuberculosis infection of cattle results in a chronic granulomatous enteritis. Clinical disease (i.e., cachexia, diarrhea, and high fecal bacterial counts) is preceded by a lengthy subclinical stage of disease. The immunologic mechanisms associated with the progression of infected cattle from subclinical to clinical disease are unclear. In this study, a cell proliferation assay was used in combination with flow cytometry to compare peripheral blood lymphocyte responses of cattle with subclinical paratuberculosis to responses of cattle with clinical paratuberculosis. B cells from cattle with subclinical disease proliferated vigorously upon stimulation with M. avium subsp. paratuberculosis antigen, with up to 12.4% of the total B cells responding. However, B cells from cattle with clinical disease did not proliferate upon antigen stimulation despite good proliferation in response to concanavalin A stimulation. In addition, these animals had high percentages of peripheral blood B cells. B cells from noninfected animals did not proliferate upon M. avium subsp. paratuberculosis antigen stimulation. Thus, it appears that B-cell proliferation is a sensitive indicator of subclinical Johne’s disease. Furthermore, the immunologic mechanisms responsible for the antigen-specific unresponsiveness of peripheral blood B cells may be significant in the eventual progression from subclinical to clinical Johne’s disease in cattle.

In rodents and humans, lymphocytes extravasate into lymph nodes via specialized paracortical venules lined with high endothelium (HEV). Sheep and other ruminants do not have morphologically defined HEV in their lymph nodes. It has been assumed that lymphocyte extravasation in these species proceeds via analogous structures; i.e., paracortical venules lined with low to medium endothelium. In this study, lymphocyte suspensions were prepared from surgically excised lymph nodes of sheep and labeled with an intracellular fluorescent dye, H33342. Labeled cells were infused intravenously back into donors, and sheep were killed at various intervals after infusion. Frozen sections of lymph nodes were examined microscopically for the location of labeled cells. Ten minutes after infusion, labeled cells were seen in the lumen of venules located in the paracortical region of the nodes. At later time points, cells were seen apparently migrating through the venule walls and in the adjacent paracortical tissue. Similar experiments were performed in which H33342-labeled murine lymphocytes were infused into syngeneic mice. When equivalent cell numbers (based on animal size) were infused, no obvious differences were seen between location and kinetics of appearance of labeled cells in lymph nodes of sheep compared to those of mice. These results indicate that lymphocyte extravasation in sheep proceeds via paracortical venules in lymph nodes. The function of these venules appears to be analogous to HEV in nonruminant species.

Experimental inoculation of neonatal immunocompetent strains of mice with Cryptosporidium parvum results in a transient, noninflammatory enteric infection. In the present study, we show that inoculation of mice deficient in alphabeta and gammadelta T cells (TCR-beta- x TCR-delta-deficient mice) with C. parvum results in persistent infection and severe inflammatory bowel disease-like lesions. The most severe lesions in these mice were in the cecum with similar yet less severe lesions in the ileum and proximal colon. The most notable aspect of the histopathology was glandular hyperplasia with abscess formation, extensive fibrosis of the lamina propria with infiltrates of predominately polymorphonuclear cells and macrophages, and a few small aggregates of B cells. Persistently infected mice also developed extensive hepatic periportal fibrosis in association with C. parvum colonization of bile ducts. Lesions observed in TCR-beta- x TCR-delta-deficient mice were markedly different than previously described lesions detected in C. parvum-infected TCR-alpha-deficient mice. Cryptosporidium parvum-infected TCR-alpha-deficient mice have extensive infiltrations of B cells, whereas TCR-beta- x TCR-delta-deficient mice had only a few small aggregates of B cells. These findings indicate that although gammadelta T cells are not necessary for induction of intestinal inflammation in C. parvum-infected alphabeta T-cell-deficient mice, their presence does alter the morphology of the ensuing lesion.

We examined patterns of lymphocyte localization in female dairy cattle following infusion of 51Cr-labeled autologous lymphocytes prepared from surgically excised mammary or ileal mesenteric lymph nodes. Labeled lymphocytes prepared from mammary lymph nodes were recovered in proportionally high numbers from mammary and prescapular lymph nodes, and in low numbers from intestinal mesenteric nodes. This pattern was observed in both heifers and lactating cows. In contrast, labeled lymphocytes prepared from ileal mesenteric lymph nodes of lactating cows were recovered in proportionally high numbers from intestinal mesenteric nodes, and in low numbers from mammary and prescapular nodes. These findings, when compared with previous results in sheep and swine, support the hypothesis that lymphocytes do not migrate efficiently between the gut and mammary gland of ruminants.