Performing Department
Microbiology and Cell Biology
Non Technical Summary
Mycoplasma ovipneumoniae is a bacterium that infects sheep and that contributes to the development of respiratory diseases. There are currently no treatments or vaccines for this pathogen. We have recently shown that Mycoplasma ovipneumoniae can form biofilms. Biofilms are communities of bacteria that stick to each other and to a surface and that therefore are very resistant to treatments. With this project, we seek to develop and test a novel Mycoplasma ovipneumoniae vaccine that targets biofilms in the respiratory tract to clear or prevent Mycoplasma ovipneumoniae infection. We hypothesize that including material from biofilms in the vaccine will lead to a strong immune response that can prevent the formation of biofilms or break up existing biofilms. In our project, we will (1) Design and produce a vaccine to Mycoplasma ovipneumoniae that incorporates biofilm components and that induces an immune response in the respiratory tract and (2) test whether a biofilm-based Mycoplasma ovipneumoniae vaccine can protect sheep from Mycoplasma ovipneumoniae infection. An effective vaccine is expected to prevent infection and improve health and productivity of lambs that are experimentally or naturally exposed to Mycoplasma ovipneumoniae. If this project is successful, farmers will finally have a tool to combat Mycoplasma ovipneumoniae infection. If we can demonstrate that incorporation of biofilm material in a Mycoplasma vaccine is effective, this approach could also be adapted for other pathogenic Mycoplasma in other agricultural species.
Animal Health Component
100%
Research Effort Categories
Basic
50%
Applied
(N/A)
Developmental
50%
Goals / Objectives
The long-term goal of our research is to develop novel strategies to eliminate Mycoplasma ovipneumoniae (M. ovi) infections from domestic sheep herds. M. ovi is a facultative pathogen that infects the respiratory tract of sheep and that is an important co-factor in ovine atypical pneumonia. With this project, we seek to develop and test a novel M. ovi vaccine that targets biofilms in the respiratory tract to clear or prevent M. ovi infection.We will test the hypothesis that an M. ovi vaccine that incorporates biofilm antigens and triggers anti-biofilm responses may protect sheep from M. ovi infection with two experimental objectives:Objective 1: Design and produce a vaccine to M. ovi that incorporates biofilm components and that induces mucosal antibody responses.Objective 2: Assess the ability of the biofilm-based M. ovi vaccine to protect sheep from M. ovi infection and improve productivity.
Project Methods
Objective 1: Design and produce a vaccine to M. ovi that incorporates biofilm components and that induces mucosal antibody responses1.a. Formulate a biofilm-based M. ovi vaccine: We will use M. ovi biofilms from 3-5 field strains of M. ovi, isolated from naturally infected MAES sheep, to generate a multivalent vaccine formulation. To generate sufficient M. ovi biofilm antigens for inoculation of sheep, we will culture the M. ovi biofilms in drip flow reactors at the CBE Medical Biofilms Laboratory. The biofilms will be homogenized, gently heat-inactivated at 56°C and sterile filtered. The preparation will then be mixed with MetaStim®, a veterinary vaccine adjuvant that acts as a lipid droplet-based delivery system, and carbopol, which activates antigen-presenting cells, promotes T cell responses, and enhances neutralizing antibody formation. A commercial vaccine for porcine M. hyopneumoniae infection that offers significant protection and results in strong mucosal IgG, IgA and IgM responses contains these two adjuvants. For quality control and standardization, we will determine the concentration of protein, carbohydrates, nucleic acids, and lipopolysaccharide (LPS) in the vaccine formulation using standard assays.1.b. Assess the safety of the adjuvanted biofilm-based M. ovi vaccine. Since we have a limited number of SPF sheep available, we first will test the safety of the vaccine in C57BL/6 mice (n=4/group). Mice will be subcutaneously (s.c.) inoculated twice over three weeks with an adjuvanted M. ovi vaccine that contains either (1) planktonic M. ovi or (2) M. ovi biofilm, each containing 5-10x106 bacteria. A similar dose of biofilm-derived M. ovi in TiterMax adjuvant induced anti-M. ovi IgG antibodies in a preliminary experiment. Mice will be observed for any adverse reactions to the vaccine, and the formulation will be adjusted if needed. If no significant adverse reactions occur and anti-M. ovi-antibody responses are induced (see 1.c. below), we will immunize four groups (n=2) of 3- to 4-month-old SPF lambs with increasing amounts of the adjuvanted, biofilm-based M. ovi vaccine. We will first immunize two lambs with the lowest dose and move on to the next group and dose only if there are no serious adverse effects. Lambs will be inoculated s.c. in the neck region twice over three weeks. Based on previous vaccine studies in sheep, doses between 50 µg and 1 mg/lamb (5x107-1x1010 bacteria) will be tested. Since higher antigen doses led to increased antibody responses in a previous M. ovi vaccine study, we will select the highest dose of the vaccine that is tolerated well by the sheep for subsequent experiments.1.c. Assess the immunogenicity of the M. ovi vaccine: To assess the immunogenicity of the M. ovi vaccine formulation, we will first analyze sera from the immunized mice, collected 3-4 weeks after the second dose, for reactivity with M. ovi. Antigen preparations from M. ovi biofilms or planktonic bacteria will be tested with the sera in an indirect ELISA. Likewise, we will assess antibody responses in the sheep from the experiment in 1.b. Over a period of three months, we will collect weekly serum samples to measure systemic antibodies, and monthly nasal wash fluids and bronchoalveolar lavages (BALs)to measure mucosal antibodies. Samples from SPF sheep inoculated with the adjuvants alone will be used for comparison. Indirect ELISA with planktonic or biofilm antigen preparations and detection antibodies that recognize ovine IgG, IgM and IgA will be used to quantify different antibody classes. To assess biofilm-specific responses, the nasal washes, BAL and sera also will be used in Western blots with antigen preparations from either planktonic M. ovi or biofilms. Cytokine arrays for pro-inflammatory, anti-inflammatory and T cell cytokines will be performed to determine the type of immune response induced, using both sera and BAL samples.To determine whether antibodies from the immunized sheep can actively disrupt existing biofilms, we will test sera, nasal washes, and BALs for anti-biofilm activity in vitro, following published protocols. We will grow M. ovi biofilms on glass bottom plates for 24-48 h in the presence of BAL, nasal washes, or sera from immunized sheep or control animals. Alternatively, we will add the BAL, nasal washes, or sera to established M. ovi biofilms to analyze whether the antibodies can induce active biofilm dispersal. We will measure biofilm mass using confocal microscopy and SYTO9 staining and will compare antibody-treated samples from immunized animals and non-immunized controls to untreated biofilms.Objective 2: Assess the ability of the biofilm-based M. ovi vaccine to protect sheep from M. ovi infection and improve productivity.2.a. Perform an M. ovi challenge experiment in specific-pathogen-free domestic lambs. We have successfully established an M. ovi infection model in our SPF sheepand will use this model to test whether the vaccine protects sheep with no prior exposure from M. ovi infection. To reduce the number of experimental animals, any immunized lambs from the experiments in 1.b/c. that have developed an antibody response will be used for a pilot experiment. Based on the pilot study, a larger group of lambs aged 2-3 months will then be used in a second, sufficiently powered challenge experiment. For experimental design, we will collaborate with the INBRE Statistical Consulting and Research Services. We will nasally challenge vaccinated and control lambs with ~5x108 M.ovi,three weeks after the second vaccine dose. Vaccine efficacy will be assessed based on M. ovi colonization in vaccinated compared to unvaccinated lambs using qPCR analysis of weekly nasal swab samples. We will also monitor clinical signs, weight gain, and feed conversion rate. Four weeks after the challenge, lambs will be euthanized, and tissue samples from the nasal cavity, tonsils, trachea, bronchi, and lungs will be harvested for analysis of M. ovi infection and histopathology. We also will measure anti-M. ovi and anti-biofilm antibody responses in sera, nasal washes, and BAL weekly after challenge (see Obj. 1).2.b. Define the impact of the M. ovi biofilm vaccine on sheep with prior M. ovi infection. Induction of anti-biofilm antibodies in response to systemic application of the biofilm-based, adjuvanted M. ovi vaccine may enable sheep with ongoing M. ovi infection to clear the bacteria. We will test this hypothesis in the MAES flock, which has a high level of natural M. ovi infection. Experimental group sizes will be determined based on the results from the challenge experiment in 2.a. For safety reasons, we will first immunize non-pregnant, 7-8 months-old ewe lambs, using the protocol described in 2.a. M. ovi infection status will be determined prior to immunization using qPCR analysis of nasal swab samples. To determine whether the M. ovi vaccine can induce sterilizing immunity, we will measure M. ovi colonization by qPCR of nasal swab samples monthly for at least three months. Impact on productivity will be assessed based on weight gains and carcass weights, where available.