Characterization of intracellular survival of Mycobacterium avium subspecies paratuberculosis in J774 murine macrophages

Mycobacterium avium subspecies paratuberculosis (M. a. ptb) is the causative agent of Johne\u27s disease, a progressive diarrheal disease of wild and domestic ruminants. Like other pathogenic species of mycobacteria, M. a. ptb is an intracellular pathogen of monocytes and monocyte-derived macrophages. In this dissertation we tested the hypothesis that M. a. ptb survives within macrophages by inhibition of acidification and maturation of the phagosomal compartment in which it resides;Chapter 1 consists of a general introduction and a literature review of recent reports concerning normal phagosomal maturation and maturation of the mycobacterial phagosome. Chapter 2 characterizes acidification of the M. a. ptb phagosomal compartment in infected J774 cells. In this study we determined that the phagosomes of M. a. ptb have diminished ability to become acidified. In Chapter 3 we evaluated maturation of the M. a. ptb phagosome by identifying two stage specific phagosomal markers, the transferrin receptor (TFR) and lysosome associated membrane protein-1 (Lamp-1). The phagosomal compartment containing M. a. ptb had reduced amounts of Lamp-1, and increased amounts of TRF, which indicates that the M. a. ptb phagosome retains characteristics of an early phagosome and does not mature into later phagosomal stages. In Chapter 4 we characterized the effects of interferon gamma (IFN-gamma) and lipopolysaccharide (LPS) on development of the M. a. ptb phagosome in infected J774 cells. We determined that IFN-gamma/LPS treated J774 cells have increased ability to cause maturation and acidification of the M. a. ptb phagosomal compartment;In this dissertation we determined that M. a. ptb survives within J774 cells by residing within a phagosomal compartment that has diminished ability to become acidified and resists normal phagosomal maturation. In addition, treatment of J774 cells with IFN-gamma/LPS increases their ability to cause acidification and maturation of the M. a. ptb phagosome

Comparative Gamma Delta T Cell Immunology: A Focus on Mycobacterial Disease in Cattle

A theme among many pathogenic mycobacterial species affecting both humans and animals is a prolonged asymptomatic or latent period that can last years to decades. The mechanisms that favor progression to active disease are not well understood. Pathogen containment is often associated with an effective cell-mediated or T-helper 1 immune profile. With certain pathogenic mycobacteria, such as Mycobacterium avium subspecies paratuberculosis, a shift to active clinical disease is associated with loss of T-helper 1 immunity and development of an ineffective humoral or T-helper 2 immune response. Recently γδ T cells have been shown to play a role early in mycobacterial infections and have been hypothesized to influence disease outcome. The purpose of this paper is to compare recent advancements in our understanding of γδ T cells in humans, cattle, and mice and to discuss roles of γδ T cells in host response to mycobacterial 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 locally injected Mycobacterium cell wall fraction in horses with sarcoids

A reformulation of Mycobacterium cell wall fraction immunotherapeutic can be used to successfully treat sarcoids in horses. Sarcoids are reported to be the most common equine skin tumors with tumor type and location influencing the choice of treatment. Wide surgical excision is curative for many tumors, but may not always be feasible. Previous studies have reported sarcoid regression after injection with mycobacterial cell wall immunotherapeutics. A new formulation of the Mycobacterium phlei cell wall fraction immunostimulant (Immunocidin® Equine) was utilized to treat cutaneous tumors in horses. Equids with skin tumors diagnosed as sarcoids were enrolled in the study. Sarcoids were injected at the initial visit with Immunocidin® Equine and subsequently at approximately 2-week intervals. Of 17 cases, 9 cases were completely resolved at the end of the study period evaluation or at time of final follow up (52.9%). Three cases were reported as improved (smaller), but not resolved (17.6%). Three cases were discontinued from the study as the respective masses were growing larger or not resolving (17.6%). One case (5.8%) with two masses had resolution of one mass, whereas the other tumor had a small regrowth 5 months after the last treatment. One case (5.8%) was lost to follow up. All cases had mild to moderate swelling of the injection site, and some cases had discharge after the second, third, or fourth injections. No serious systemic side effects or complications were encountered during the study

Disseminated \u3ci\u3eLeishmania infantum\u3c/i\u3e infection in two sibling foxhounds due to possible vertical transmission

Two sibling foxhounds born to a Leishmania seropositive bitch were presented after testing seropositive for Leishmania. Leishmania infantum infection was detected via histopathology, culture, and quantitative polymerase chain reaction (q-PCR). This is the first report of natural infection with Leishmania infantum with the possibility for vertical transmission in North America. Infection disséminée à Leishmania infantum chez deux chiots Fox hound d’une même portée reliée possiblement à une transmission verticale. Deux chiots Fox hound d’une même portée nés d’une mère séropositive à Leishmania ont été présentés après un contrôle sérologique positif. Une infection à Leishmania infantum a été détectée par histopathologie, culture et amplification en chaîne par polymérase quantitative (ACP-q). Il s’agit du premier rapport d’infection naturelle par Leishmania infantum possiblement relié à une transmission verticale en Amérique du Nord

Prevention of Mycobacterium avium Subspecies paratuberculosis (MAP) Infection in Balb/c mice by Feeding Lactobacillus acidophilus Strain NP-51®

The immune responses of 390 BALB/c mice fed the probiotic Lactobacillus acidophilus strain NP51 ® and infected with Mycobacterium avium subspecies paratuberculosis (MAP) were evaluated in a 6-month trial. Mice were randomized to nine treatment groups that fed either viable- or heat-killed NP51 and inoculated with either viable- or heatkilled MAP or sterile phosphate-buffered saline. Feeding the NP51 resulted in higher numbers of T lymphocytes in the spleen including the CD8 + cytotoxic T lymphocytes. In addition, feeding the NP51 lowered the number of immune suppressive T regulatory cells CD4 + CD25 + and CD8 + CD25 + cells in the spleen. Additionally, feeding the NP51 resulted in higher concentration of interferon-gamma in the supernatant of splenocytes cultured in vitro. These results suggest that feeding the NP51 to BALB/c mice might prevent the progression of MAP infection in mice

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

Helicobacter bilis Infection Alters Mucosal Bacteria and Modulates Colitis Development in Defined Microbiota Mice

Background: Helicobacter bilis infection of C3H/HeN mice harboring the altered Schaedler flora (ASF) triggers progressive immune responsiveness and the development of colitis. We sought to investigate temporal alterations in community structure of a defined (ASF-colonized) microbiota in normal and inflamed murine intestines and to correlate microbiota changes to histopathologic lesions. Methods: The colonic mucosal microbiota of healthy mice and ASF mice colonized with H. bilis for 3, 6, or 12 weeks were investigated by fluorescence in situ hybridization targeting the 16S ribosomal RNA genes of total bacteria, group-specific organisms, and individual ASF bacterial species. Microbial profiling of ASF and H. bilis abundance was performed on cecal contents. Results: Helicobacter bilis–colonized mice developed colitis associated with temporal changes in composition and spatial distribution of the mucosal microbiota. The number of total bacteria, ASF519, and helicobacter-positive bacteria were increased (P , 0.05), whereas ASF360/361-positive bacteria were decreased (P , 0.05) versus controls. Adherent biofilms in colitic mice were most often (P , 0.05) composed of total bacteria, ASF457, and H. bilis. Total numbers of ASF519 and H. bilis bacteria were positively correlated (P ¼ 0.03, r ¼ 0.39 and P , 0.0001, r ¼ 0.73), and total numbers of ASF360/361 bacteria were negatively correlated (P ¼ 0.003, r ¼ 20.53) to histopathologic score. Differences in cecal abundance of ASF members were not observed. Conclusions: Altered community structure with murine colitis is characterized by distinct ASF bacteria that interact with the colonic mucosa, by formation of an isolating interlaced layer, by attachment, or by invasion, and this interaction is differentially expressed over time