Mycoplasma bovis

Investigator:

• Jose Perez-Casal

Overview:

Mycoplasma bovis is known to cause chronic bovine pneumonia and poly-arthritis syndrome (CPPS) in feedlot cattle. These infections are responsible for considerable economic losses, estimated for the U.S. at $32 million per year. Antibiotic treatment is costly and not very effective, leaving vaccination as an alternative approach. While there are bacterin-based commercial vaccines, they are prepared from a limited number of strains, and since M. bovis shows a high degree of antigenic variation, these vaccines might not protect against all M. bovis isolates. We propose to develop a vaccine for calves that can be administered at branding time, followed by a boost vaccination before or at the time of arrival at the feedlot since it is the young calf that is of highest risk of exposure to M. bovis. Our interest also resides on the study of the interaction between M. bovis and host cells. We are using kinome array technology, developed at VIDO by Dr. Scott Napper to analyze innate immune phosphorylation pathways that are modulated by M. bovis. We are part of an international group funded by The Canadian International Food Security Research Fund (CIFSRF) to develop recombinant vaccines against Mycoplasma mycoides, the causative agent of Contagious Bovine Pleuropneumonia (CBPP), a communicable disease in North America. This project will use reverse vaccinology approaches to identify targets for vaccines to be used in different countries in Africa where the losses due to CBPP cause severe burden to the local economies.

Background:

Reports in the literature indicate that M. bovis can be found in the cytoplasm of cells where it can evade the immune response of the host. Thus, a vaccine is required that induces both humoral and cell-mediated immune responses (CMIR) which are needed for intracellular pathogens. Work by other researchers at VIDO showed that that vaccination with plasmid DNA elicits strong and long-lasting cell-mediated immune responses (CMIR) in young calves. Thus, we are planning to use a plasmid DNA carrying the gene encoding GAPDH alone or in combination with the recombinant GAPDH protein in our vaccine trials.  Since our target is the young calf, we plan to use vaccine adjuvants that are effective in early life such as host-defence peptides (HDPs) and synthetic oligonucleotides (ODNs) known as CpG. Recent studies have shown that in combination, HDPs and CpG possess a synergistic effect, eliciting strong CMIR to weak antigens and promoting prolonged duration of immune responses following a single injection. Since HDP also have antimicrobial properties we plan to test for the ability of these HDP to kill a culture of M. bovis. As part of our current research on M. bovis vaccines, we developed a challenge model where young calves (3-4 weeks of age) from farms with no history of M. bovis infections were separated from their dams at 4-7 days of age, hand-raised, kept in isolation and challenged with a strain of M. bovis. While this model is useful for initial testing of experimental vaccines, it is not representative of what happens in the feedlots. Animals arriving at feedlots are older, sourced from large numbers of cow-calf operations and auction marts and frequently are exposed to other pathogens such as Bovine Virus Diarrhoea Virus (BVDV) and Bovine Herpes virus (BHV-1), pathogens that are strongly associated with the occurrence of M. bovis. 

We and others reported that M. bovis is capable of inhibiting bovine peripheral-blood mononuclear cell (BPBMC) proliferation [2]. In a recent study, we determined that the inhibitory factor is found in culture supernatants and that M. bovis is capable of invading BPBMC and erythrocytes with no apparent loss of cell viability. Moreover, we determined that M. bovis is capable of delaying programmed cell death (Apoptosis) in BPBMC which could help the bacterium to evade immune responses and spread to tissues in the host.

Contagious Bovine Pleuropneumonia is a disease caused by Mycoplasma mycoides. While this disease has been eradicated from North America in the late 1800’s, it is still a serious problem in several African countries. Not only CBPP causes severe economic losses to the local economies but also significantly reduces animal trade within Africa and also with other countries. There is a need for new recombinant vaccines that do not harm the host and we plan to use a proteomic approach to identify targets for these vaccines.

Objectives:

• To conduct in-vitro assays using bovine monocytes and alveolar macrophages to study host-pathogen interactions with a focus on the role that M. bovis plays in subverting innate immune responses in the host. 
• To identify the M. bovis products that modulates BPBMC proliferation
• To test experimental vaccines against M. bovis by using our recently developed BHV-1-M. bovis co-infection challenge-model for feedlot cattle.
• To use reverse vaccinology for identification of M. mycoides vaccine targets and conduct animal trials with the purified recombinant proteins to select the best antigens for a new vaccine against this important pathogen.

Progress:

To date, we have identified three HDP that elicited cytokine production by BPBMC. We have constructed  three chimeric proteins composed of the M. bovis GAPDH protein and HDP and shown that these chimeras retain the properties of the individual components i.e. GAPDH and antimicrobial activities[3].

We found that M. bovis is capable of invading B cells, monocytes, γ-δ T cells, dendritic cells, NK-cells, cytotoxic T cells and T-helper cells without apparently affecting their viability. Also, we found M. bovis inside bovine erythrocytes.  This may help to explain the inhibition of immune responses observed after incubation of M. bovis with BPBMC and the route of spread from the lungs to other tissues in the body.

We are currently examining the interaction between M. bovis and monocytes and alveolar macrophages using kinome arrays. These assays will allow us to identify innate immune pathways affected by M. bovis and the findings could be used to develop intervention strategies to combat M. bovis diseases. We have identified several M. bovis antigens that could be used as targets for vaccine development and plan to test these antigens using our M. bovis co-infection challenge model in combination with novel adjuvant formulations developed at VIDO. In this model, a previous exposure of feedlot-age calves to BHV-1 followed by an intra-nasal challenge with M. bovis results in clinical disease consistent with CPPS.