Tracy Prysliak

Invasion of Bovine Peripheral Blood Mononuclear Cells and Erythrocytes by Mycoplasma bovis

ABSTRACT Mycoplasma bovis is a small, cell wall-less bacterium that contributes to a number of chronic inflammatory diseases in both dairy and feedlot cattle, including mastitis and bronchopneumonia. Numerous reports have implicated M. bovis in the activation of the immune system, while at the same time inhibiting immune cell proliferation. However, it is unknown whether the specific immune-cell population M. bovis is capable of attaching to and potentially invading. Here, we demonstrate that incubation of M. bovis Mb1 with bovine peripheral blood mononuclear cells (PBMC) resulted in a significant reduction in their proliferative responses while still remaining viable and capable of gamma interferon secretion. Furthermore, we show that M. bovis Mb1 can be found intracellularly (suggesting a role for either phagocytosis or attachment/invasion) in a number of select bovine PBMC populations (T cells, B cells, monocytes, γδ T cells, dendritic cells, NK cells, cytotoxic T cells, and T-helper cells), as well as red blood cells, albeit it at a significantly lower proportion. M. bovis Mb1 appeared to display three main patterns of intracellular staining: diffuse staining, an association with the intracellular side of the cell membrane, and punctate/vacuole-like staining. The invasion of circulating immune cells and erythrocytes could play an important role in disease pathogenesis by aiding the transport of M. bovis from the lungs to other sites.

Vaccination of feedlot cattle with extracts and membrane fractions from two Mycoplasma bovis isolates results in strong humoral immune responses but does not protect against an experimental challenge.

Mycoplasma bovis is one of the most significant contributors to the bovine respiratory syndrome (BRD) that causes major losses in feedlot and dairy farms. Current experimental vaccines against M. bovis are ineffective and in some cases seem to enhance disease. Experimental infection with M. bovis induces a predominantly Th2 response and high levels of IgG1, which is an inferior opsonin and hence lacks protective capacity. In an attempt to induce a balanced (Th1/Th2) immune response, we have used CpG ODN 2007 as an adjuvant in a trial involving vaccination of cattle with M. bovis total extracts and/or membrane fractions and subsequent intranasal inoculation with an infective dose of M. bovis prepared from two different clinical isolates. Significant IgG1 serum responses were observed against both, extracts and fractions while IgG2 responses were significant against the extracts only. Proliferation of peripheral blood mononuclear cells (PBMC) after incubation with M. bovis cells was only observed in post-challenge samples of cattle vaccinated with both extracts and fractions but not in samples of cattle immunized with the membrane fractions alone. All groups showed transient weight losses and increased temperatures however, there were no significant differences in clinical parameters and survival rates between the groups.

Vaccination with recombinant Mycoplasma bovis GAPDH results in a strong humoral immune response but does not protect feedlot cattle from an experimental challenge with M. bovis

Mycoplasma bovis continues to cause significant disease in feedlots and dairy farms. The ability of the micro-organism to evade the immune system of the host combined with the lack of effective vaccines makes this disease difficult to control. Bacterin-based vaccines have not been successful in field trials and in some cases enhance the disease. In an attempt to develop a sub-unit vaccine, we used the conserved M. bovis glyceraldehyde-3-phosphate (GAPDH) protein in combination with a protein extract prepared from three M. bovis isolates to immunize feedlot animals. After challenge with a combination of three M. bovis isolates, there were differences in the proportion of weight loss between the control and vaccinated groups but no differences in rectal temperature and survival rate in all the groups. In addition, there were no significant differences between the proportions of lungs lesions in all the groups despite the percentages of lesions being higher in the vaccinated groups. These findings indicate that the M. bovis GAPDH protein is not a suitable antigen for a vaccine against this pathogen.

Protein chimeras containing the Mycoplasma bovis GAPDH protein and bovine host-defence peptides retain the properties of the individual components.

Besides the well characterized role in glycolysis, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been implicated in virulence of pathogenic micro-organisms and because of its cell surface location, it has been shown to act as an adhesin for colonization of tissue surfaces both for pathogenic and non-pathogenic normal microflora. These novel properties of GAPDH make this protein a target for studies in pathogenesis and a candidate for vaccine development against several diseases. Previously, we have isolated the GAPDH protein of Mycoplasma bovis and we are currently using this protein as a test antigen to develop a vaccine to protect feedlot animals from M. bovis-related diseases. As part of our vaccine studies, we are testing several novel immune modulators, some of which are host-defence peptides (HDP). HDP are small protein molecules that are part of the innate immune system of the host possess antimicrobial activities and can act as adjuvants. These novel compounds have been used as part of chimeric proteins composed of viral antigens fused to HDP and these chimeras were found to promote immune responses. The first step in the use of the M. bovis GAPDH protein and HDP as components of a vaccine was to construct M. bovis GAPDH-HDP chimeric proteins. The three M. bovis GAPDH-HDP chimeric proteins constructed here: GAPDH-BMAP28 (sGap-M), GAPDH-indolicidin (sGap-I), and GAPDH-TAP (Gap-T) retained properties associated with the individual components, namely GAPDH enzymatic and HDP antimicrobial activities.

Role of GapC in the pathogenesis of Staphylococcus aureus.

Staphylococcus aureus is recognized worldwide as a major pathogen causing clinical or subclinical intramammary infections in lactating cows, sheep and goats. S. aureus produces a wide arsenal of cell surface and extracellular proteins involved in virulence. Among these are two conserved proteins with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity named glyceraldehyde-3-phosphate dehydrogenase-B (GapB) and -C (GapC). In this study, we used the S. aureus wild type strain RN6390 and its isogenic gapC mutant H330 in in vitro and in vivo studies and determined that the S. aureus GapC protein plays a role on adherence to and internalization into bovine mammary epithelial (MAC-T) cells. In addition, we found that S. aureus H330 did not caused mastitis after an experimental infection of ovine mammary glands. Together, these results show that GapC is important in the pathogenesis of S. aureus mastitis.

Mycoplasma bovis isolates recovered from cattle and bison (Bison bison) show differential in vitro effects on PBMC proliferation, alveolar macrophage apoptosis and invasion of epithelial and immune cells.

In the last few years, several outbreaks of pneumonia, systemically disseminated infection, and high mortality associated with Mycoplasma bovis (M. bovis) in North American bison (Bison bison) have been reported in Alberta, Manitoba, Saskatchewan, Nebraska, New Mexico, Montana, North Dakota, and Kansas. M. bovis causes Chronic Pneumonia and Polyarthritis Syndrome (CPPS) in young, stressed calves in intensively-managed feedlots. M. bovis is not classified as a primary pathogen in cattle, but in bison it appears to be a primary causative agent with rapid progression of disease with fatal outcomes and an average 20% mature herd mortality. Thus, there is a possibility that M. bovis isolates from cattle and bison differ in their pathogenicity. Hence, we decided to compare selected cattle isolates to several bison isolates obtained from clinical cases. We show differences in modulation of PBMC proliferation, invasion of trachea and lung epithelial cells, along with modulation of apoptosis and survival in alveolar macrophages. We concluded that some bison isolates showed less inhibition of cattle and bison PBMC proliferation, were not able to suppress alveolar macrophage apoptosis as efficiently as cattle isolates, and were more or less invasive than the cattle isolate in various cells. These findings provide evidence about the differential properties of M. bovis isolated from the two species and has helped in the selection of bison isolates for genomic sequencing.

Effect of Mycoplasma bovis on bovine neutrophils

Mycoplasma bovis (M. bovis) is a small bacterium that lacks a cell wall. M. bovis infection results in chronic pneumonia and polyarthritis syndrome (CPPS), otitis media, conjunctivitis, and meningitis in feedlot cattle and mastitis in dairy cattle. Numerous studies of peripheral mononuclear cells (PBMC) indicate that M. bovis evades host immunity through targeted effects on immune cell activity, including inhibition of effector function and simultaneous aberrant activation of immune cell activity that has no effect on protection against the bacterium. Few studies have addressed the interaction between M. bovis and neutrophils, one of the most important cell subsets of innate immunity. We hypothesized that M. bovis modifies specific neutrophil activities to support its persistence and systemic dissemination. In this study, we demonstrate that M. bovis enhances neutrophil apoptosis, stimulates production of pro-inflammatory cytokines, IL-12 and TNF-α, inhibits production of nitric oxide (NO) but augments elastase release. We also show that IL-17 an inflammatory cytokine produced by Th-17 cells does not enhance the capacity of neutrophils to destroy M. bovis. These findings present novel mechanisms of mycoplasma evasion of host innate immunity and provide potential opportunities for immuno-therapeutic interventions.

Immune responses to Mycoplasma bovis proteins formulated with different adjuvants.

Most vaccines for protection against Mycoplasma bovis disease are made of bacterins, and they offer varying degrees of protection. Our focus is on the development of a subunit-based protective vaccine, and to that end, we have identified 10 novel vaccine candidates. After formulation of these candidates with TriAdj, an experimental tri-component novel vaccine adjuvant developed at VIDO-InterVac, we measured humoral and cell-mediated immune responses in vaccinated animals. In addition, we compared the immune responses after formulation with TriAdj with the responses measured in animals vaccinated with a mix of a commercial adjuvant (Emulsigen™) and 2 of the components of the TriAdj, namely polyinosinic:polycytidylic acid (poly I:C) and the cationic innate defense regulator (IDR) peptide 1002 (VQRWLIVWRIRK). In this latter trial, we detected significant IgG1 humoral immune responses to 8 out of 10 M. bovis proteins, and IgG2 responses to 7 out of 10 proteins. Thus, we concluded that the commercial adjuvant formulated with poly I:C and the IDR peptide 1002 is the best formulation for the experimental vaccine.

Status of the development of a vaccine against Mycoplasma bovis

Mycoplasma bovis is an important pathogen of cattle and, despite numerous efforts an effective vaccine for control of the disease it causes remains elusive. Although we now know more about the biology of this pathogen, information is lacking about appropriate protective antigens, the type of immune response that confers protection and adjuvants selection. The use of conserved recombinant proteins, selected using in silico approaches, as components of a vaccine may be a better choice over bacterin-based vaccines due to the limited protection afforded by them and adverse reactions caused by them. More studies are needed on the characterization of host-pathogen interactions and to elucidate M. bovis products modulating these interactions. These products could be the basis for development of vaccines to control M. bovis infections in dairy farms and feedlots.

Analysis of immune responses to recombinant proteins from strains of Mycoplasma mycoides subsp. mycoides, the causative agent of contagious bovine pleuropneumonia.

Current contagious bovine pleuropneumonia (CBPP) vaccines are based on live-attenuated strains of Mycoplasma mycoides subsp. mycoides (Mmm). These vaccines have shortcomings in terms of efficacy, duration of immunity and in some cases show severe side effects at the inoculation site; hence the need to develop new vaccines to combat the disease. Reverse vaccinology approaches were used and identified 66 candidate Mycoplasma proteins using available Mmm genome data. These proteins were ranked by their ability to be recognized by serum from CBPP-positive cattle and thereafter used to inoculate naïve cattle. We report here the inoculation of cattle with recombinant proteins and the subsequent humoral and T-cell-mediated immune responses to these proteins and conclude that a subset of these proteins are candidate molecules for recombinant protein-based subunit vaccines for CBPP control.