Predicting the Role of IL-10 in the Regulation of the Adaptive Immune Responses in Mycobacterium avium Subsp. paratuberculosis Infections Using Mathematical Models

Mycobacterium avium subsp. paratuberculosis (MAP) is an intracellular bacterial pathogen that causes Johne’s disease (JD) in cattle and other animals. The hallmark of MAP infection in the early stages is a strong protective cell-mediated immune response (Th1-type), characterized by antigen-specific γ-interferon (IFN-γ). The Th1 response wanes with disease progression and is supplanted by a non-protective humoral immune response (Th2-type). Interleukin-10 (IL-10) is believed to play a critical role in the regulation of host immune responses to MAP infection and potentially orchestrate the reversal of Th1/Th2 immune dominance during disease progression. However, how its role correlates with MAP infection remains to be completely deciphered. We developed mathematical models to explain probable mechanisms for IL-10 involvement in MAP infection. We tested our models with IL-4, IL-10, IFN-γ, and MAP fecal shedding data collected from calves that were experimentally infected and followed over a period of 360 days in the study of Stabel and Robbe-Austerman (2011). Our models predicted that IL-10 can have different roles during MAP infection, (i) it can suppress the Th1 expression, (ii) can enhance Th2 (IL-4) expression, and (iii) can suppress the Th1 expression in synergy with IL-4. In these predicted roles, suppression of Th1 responses was correlated with increased number of MAP. We also predicted that Th1-mediated responses (IFN-γ) can lead to high expression of IL-10 and that infection burden regulates Th2 suppression by the Th1 response. Our models highlight areas where more experimental data is required to refine our model assumptions, and further test and investigate the role of IL-10 in MAP infection

Transcriptional Profiling of Ileocecal Valve of Holstein Dairy Cows Infected with Mycobacterium avium subsp. Paratuberculosis

Johne’s disease is a chronic infection of the small intestine caused by Mycobacterium avium subspecies paratuberculosis (MAP), an intracellular bacterium. The events of pathogen survival within the host cell(s), chronic inflammation and the progression from asymptomatic subclinical stage to an advanced clinical stage of infection, are poorly understood. This study examines gene expression in the ileocecal valve (ICV) of Holstein dairy cows at different stages of MAP infection. The ICV is known to be a primary site of MAP colonization and pro-vides an ideal location to identify genes that are relevant to the progression of this disease. RNA was prepared from ICV tissues and RNA-Seq was used to compare gene transcription between clinical, subclinical, and uninfected control animals. Interpretation of the gene expression data was performed using pathway analysis and gene ontology categories containing multiple differentially expressed genes. Results demonstrated that many of the pathways that had strong differential gene expression between uninfected control and clinical cows were related to the immune system, such as the T- and B-cell receptor signaling, apoptosis, NOD-like receptor signaling, and leukocyte transendothelial migration pathways. In contrast, the comparison of gene transcription between control and subclinical cows identified pathways that were primarily involved in metabolism. The results from the comparison between clinical and subclinical animals indicate recruitment of neutrophils, up-regulation of lysosomal peptidases, increase in immune cell transendothelial migration, and modifications of the extracelluar matrix. This study provides important insight into how cattle respond to a natural MAP infection at the gene transcription level within a key target tissue for infection

Comparison of Sheep, Goats, and Calves as Infection Models for Mycobacterium avium subsp. paratuberculosis

Animal infection models to study Mycobacterium avium subsp. paratuberculosis (MAP) infection are useful for evaluating the efficacy of vaccines and other therapeutics for the prevention or treatment of infection. The goal of the present study was to compare smaller ruminants, sheep and goats, with calves as infection models. Neonatal sheep, goats, and calves (n = 4) received 109 cfu of a cattle isolate of MAP in milk replacer on days 0, 3 and 6 in a 12-month study and sampled monthly thereafter. Results demonstrated a robust antigen-specific IFN-γ response at 90 days post-inoculation for sheep and goats, with lower responses noted for calves. By 360 days, IFN-γ responses were 50 and 82% higher for calves than for goats and sheep, respectively. Although MAP- specific antibody responses were first observed in sheep at 90 days, calves had higher antibody responses throughout the remainder of the study. Following pass-through shedding on day 7, fecal shedding was fairly negligible across treatments but remained higher for calves throughout the study. Colonization of tissues was variable within treatment group and was higher for calves and sheep for the majority of tissues. Upon antigen stimulation of PBMCs, higher populations of CD4 + T cells cells and lower populations of γδ TCR + and NK cells were observed for goats and calves compared to sheep. Relative gene expression of IL-4, IL-12, and IL-17 in PBMCs was higher in goats, corresponding to lower tissue colonization with MAP. These data suggest that ruminant species are fairly comparable as infection models for MAP, but discrete differences in host responses to MAP infection exist between species

Evaluation of protection in a mouse model after vaccination with Mycobacterium avium subsp. paratuberculois protein cocktails

Whole-cell vaccines successfully reduce signs of clinical disease and fecal shedding of Mycobacterium avium subsp. paratuberculosis (MAP), however, these vaccines have some limitations. The present study was conducted to identify MAP proteins that might be candidates for the development of an improved vaccine. MAP proteins were screened for immunogenicity in naturally infected cattle and selected based upon reactivity in the interferon- (IFN-) and Western blot assays. Proteins (MAP1087, MAP1204, MAP1272c, and MAP2077c) were arrayed into 4 overlapping cocktails containing 3 proteins each. The efficacy of the proteins within these cocktails as vaccine candidates was evaluated by subcutaneous immunization of mice, followed by challenge with live, virulent MAP. All MAP protein cocktails significantly reduced the recovery of live MAP from the ileum, while cocktails 1 and 3 reduced colonization in the liver. No significant differences were seen in the mesenteric lymph node or spleen, however, cocktail 1 reduced viable MAP in the mesenteric lymph node compared to other treatments. Stimulation of splenocytes upregulated antigen-specific IFN- and IL-23 secretion in all treatment groups, regardless of vaccination. Interestingly, IL-4 was moderately downregulated for vaccinates compared to control infected mice. An increase in total CD25 expression was noted for 3 of the 4 vaccinate groups upon stimulation of splenocytes with a whole-cell sonicate of MAP, with this effect becoming more significant within CD4CD25+ and CD8CD25+ subpopulations. The present study demonstrated that MAP proteins are useful as vaccine candidates to reduce MAP tissue burden

Phenotypes of macrophages present in the intestine are impacted by stage of disease in cattle naturally infected with Mycobacterium avium subsp. paratuberculosis

Macrophages play an important role in the host immune response to Mycobacterium avium subsp. paratuberculosis (MAP) infection, however, MAP is able to disrupt normal macro- phage functions to avoid destruction. It is unclear whether the phenotypes of macrophages present in the target tissue play a role in the inability to clear MAP infection. The aim of this study was to identify macrophage phenotypes (host defense or resolution and repair) present within the bovine ileum of naturally infected cattle, as well as to ascertain abundance of each macrophage phenotype present during different stages of MAP infection. Immunofluo- rescent (IF) labeling was performed on frozen bovine mid-ileal tissue sections collected from 28 Holstein dairy cows. Comprehensive IF staining for cytokines, such as IFN-γ, IL- 1Ra, IL-1β, IL-10, TGF-β, TNF-α, and uNOS, along with markers such as CD163, CD206, and TLR4, served to define the macrophage phenotypes. Overall, cows in the clinical stage of disease demonstrated significantly higher numbers of resolution and repair macrophages and lower numbers of host defense macrophages in the ileal tissue. Interestingly, subclinically affected cows with asymptomatic disease had a nearly equal ratio of host defense and resolution and repair macrophage phenotypes, whereas macrophage phenotype was skewed to a host defense macrophage in the tissues of the control noninfected cows. The preponderance of M2-like resolution and repair phenotype for macrophages in the tissues of cows with clinical disease would explain why the host fails to control and/or clear the infection, leading to a higher MAP burden. The results of the current study offer insight into the disparate macrophage phenotypes present in the bovine ileum during different stages of infection

Genetic markers for screening animals for improved disease resistance (NRAMP)

A method for determining improved innate immunity, disease resistance or performance in animals is disclosed. The method involves assays for a genetic differences in the NRAMP1 gene of the animal which is associated with superior disease resistance. Novel NRAMP1 sequence, assays, and compositions for identifying the presence of absence of these alleles are provided

Early Epithelial Invasion by Salmonella enterica Serovar Typhimurium DT104 in the Swine Ileum

Salmonella enterica serovar Typhimurium is an important intestinal pathogen in swine. This study was performed to document the early cellular invasion of Salmonellaserovar Typhimurium in swine ileum. Ileal gut-loops were surgically prepared in ten 4- to 5-week-old mixed-breed pigs and inoculated for 0-60 minutes. Loops were harvested and prepared for both scanning and transmission electron microscopy (SEM and TEM, respectively). Preferential bacterial adherence to microfold cells (M cells) was seen within 5 minutes, and by 10 minutes bacterial invasion of the apical membrane was seen in M cells, goblet cells, and enterocytes. This multicellular invasion was observed throughout the course of infection. In addition, SEM revealed a specific affinity of Salmonella serovar Typhimurium to sites of cell extrusion. Using TEM, bacteria in these areas were focused in the crevices formed by the extruding cell and the adjacent cells and in the cytoplasm immediately beneath the extruding cell. Our results suggest that early cellular invasion by Salmonella serovar Typhimurium is nonspecific and rapid in swine. Furthermore, the combination of SEM and TEM data suggests that Salmonella serovar Typhimurium may use sites of cell extrusion as an additional mechanism for early invasion

Quantification of Macrophages and Mycobacterium avium Subsp. paratuberculosis in Bovine Intestinal Tissue During Different Stages of Johne’s Disease

Johne’s disease is an enteric disease caused by the intracellular pathogen Mycobacterium avium subsp. paratuberculosis (MAP). Upon ingestion of MAP, it is translocated across the intestinal epithelium and may be killed by intestinal macrophages, or depending on the bacterial burden and immunological status of the animal, MAP may thwart innate defense mechanisms and persist within the macrophage. This study aimed to determine the numbers of macrophages and MAP present in bovine midileal tissue during different stages of infection. Immunofluorescent (IF) labeling was performed on frozen bovine midileal intestinal tissue collected from 28 Holstein dairy cows. The number of macrophages in midileal tissue sections was higher for clinically affected cows, followed by subclinically affected cows and then uninfected control cows. Macrophages were present throughout the tissue sections in clinical cows, including the tunica muscularis, submucosa, and the lamina propria around the crypts and in the villous tips, with progressively fewer macrophages in subclinically affected and control cows. Clinically affected cows also demonstrated significantly higher numbers of MAP and higher numbers of macrophages with intracellular MAP compared to subclinically affected cows. MAP IF labeling was present within the submucosa and lamina propria around the crypts, progressing into the villous tips in some clinically affected cows. Our findings indicate that number of macrophages increases with progression of infection, but a significant number of the macrophages present in the midileum are not associated with MAP