Objective 1 will focus on understanding the epidemiology and transmission of Mycobacterial diseases in animals. To accomplish our overall objective of developing a better understanding of the epidemiology and transmission of JD and TB, we propose studies that include:
(i) Continued development of mathematical models of JD and TB transmission dynamics, including within-host, between individuals, within and between domesticated dairy and beef herds and wildlife, as well as on an ecological scale. For example, several investigators have initiated the process of development of mathematical models for JD and TB (2-4) and we will continue the process with studies such as estimating the performance of JD vaccines, defining the impact of wildlife infection on JD and TB dynamics, analyzing the spread of JD and TB through cattle trading networks, and finding economically optimal JD and TB control strategies. Examples of the types of investigations that will be carried out are presented in (5-7).
(ii) Characterization of herd and environmental distribution of specific genotypes of M. paratuberculosis and M. bovis using state-of-the-art methods for strain differentiation using simple sequence repeats and or single nucleotide based typing approaches and applying this knowledge to characterize the genetic diversity and molecular epidemiology of M. paratuberculosis and M. bovis infections;
(iii) Delineation of mycobacterial disease transmission dynamics, including M. paratuberculosis transmission within calf-rearing systems, risk of M. paratuberculosis transmission from infected dams to daughters, and risk of M. paratuberculosis infection associated with ‘super-shedders’ and calf-to-calf transmission;
(iv) Clarification and delineation of critical management practices for control, prevention, and eradication of mycobacterial diseases; and,
(v) Identification and optimization of surveillance methods and strategies.
Taken together, these studies will significantly advance our understanding of the epidemiology and transmission dynamics of mycobacterial diseases of animals.
Objective 2 will seek to develop and implement new generations of diagnostic tests for JD and TB. Improved methods for the rapid, specific, sensitive, and cost-efficient diagnosis of JD or TB infected remain a major priority. Hence, as part of this multi-state initiative, we anticipate carrying out investigations that include:
(i) Development of methods for the early detection of M. paratuberculosis and M. bovis infected animals, including newer generations of molecular, serological and microbiological assays with greater sensitivity, specificity, speed, and or ease-of-use, by using state-of-the art molecular biological, immunological, and materials science and engineering methods and approaches; and,
(ii) Development of resources for validation and standardization of diagnostic assays, including well-accessioned biological sample collections (strains, tissue, clinical samples, etc.), and processes to make these accessible to the scientific community.
Together, these studies and efforts will facilitate the development, validation, and implementation of the next-generation of improved diagnostic tests for mycobacterial diseases of animals.
Objective 3 will focus on improving our understanding of biology and pathogenesis of Mycobacterial diseases of animals, as well as the host response to infection. Our understanding of the basic biology and mechanisms of pathogenesis of M. paratuberculosis and M. bovis is far from complete. It is well recognized that the ability to identify the route of invasion and the host-pathogen interactions at a molecular level is important for the future development of strategies to prevent infections or to limit the spread of the infection. Similarly, the elucidation of gene products specific to in vivo growth holds great promise in identifying new antigens for diagnostics or vaccine development, as well as products essential to pathogenesis.
Hence, as part of the proposed multi-state initiative, we envision studies of the basic biology of the causative organisms of JD and TB and their interaction with the host. Specifically, we anticipate studies that will employ state-of-the art microbiological, molecular biology, genomic, proteomic, metabolomic, immunology, and or bioinformatic approaches to carry out studies that include:
(i) Investigations into the basic mechanisms of pathogen invasion of host cells and tissue using state-of the art methods in mycobacteriology, cell biology, and genomics;
(ii) Identification of mycobacterial genes and proteins whose inactivation or alternated expression results in reduced virulence. This will be accomplished by screening large libraries of mutants, as well as by characterizing these mutant strains using state-of-the art genomics and proteomics based methods and will also lead to the identification of genes associated with the ability of the pathogen to survive in the host as markers for virulence and pathogenicity; and,
(iii) Characterization of the microbial factors that contribute to the innate and adaptive immune response using sophisticated in vitro cellular immunologic assays and animal models of infection.
(iv) Exploitation of knowledge from immune response studies to create new methods of diagnosis.
Taken together, we anticipate that these investigations will reveal important insights on the basic biology of the causative organisms of JD and TB and their interaction with their hosts.
Objective 4 will focus on the evaluation and development of new generations of vaccines for JD and TB. It is well recognized that defining the host genetic, cellular and molecular events associated with susceptibility to JD and TB is essential for the development of candidate vaccines and host genetic selection for resistance. For TB in particular, the experience in the UK and elsewhere have shown that traditional test/slaughter and abattoir inspection campaigns fail to control the spread of bovine TB (bTB), most likely due to the presence of a wildlife reservoir. Vaccine research must become a priority. Similarly, in the US where a wildlife reservoir exists, control efforts have not eradicated bTB and are unlikely to do so. Hence, the development of a vaccine against bTB is required to control disease. Under the auspices of this multi-state initiative, we envision projects that will seek to develop candidate vaccines, identify genes and markers associated with susceptibility of animals to mycobacterial infection, and define the cellular and molecular events associated with development of immune responses to M. paratuberculosis and M. bovis in cattle. Specifically, we anticipate the development of projects that will:
(i) Analyze the early immune response to infection as well as the host response to animals at different stages of disease using well-characterized in vitro models and animal experimentation;
(ii) Develop and validate animal models for vaccine development;
(iii) Identify genetic markers for susceptibility to infection in cattle using genome wide association studies with well-defined resource populations. A combination of candidate gene identification with whole genome SNP typing promises to rapidly identify a set of markers that could be used to select for resistance to disease caused by mycobacteria;
(iv) Compare the efficacy of candidate vaccines in animal models of infection. We hypothesize that live attenuated vaccines are likely to elicit a protective response superior to the response elicited by currently available killed vaccines. However, it will be essential to develop vaccine candidates that are able to differentiate vaccinated from naturally infected animals. To test this hypothesis, we anticipate studies that include: (a) use of flow cytometry, long-oligo microarrays, and real time RT-PCR to compare immune responses elicited by candidate mutant vaccines; (b) Determine if mutant vaccines elicit development of effector memory CD4 and/or CD8 T cells that kill infected autologous macrophages or arrest replication of intracellular bacteria; and, (c) Determine if animal immunized with mutant vaccines are protected against challenge;
(v) Evaluate the ability of recombinant or vector expressed proteins and mycobacterial lipids to elicit effector T cells with the capacity to kill infected macrophages or arrest replication of intracellular bacteria. The working hypothesis is that modification of mycobacterial antigens by attachment of Trojan peptides will selectively enhance development of long-lived memory CD4 and/or CD8 effector T cells and may be suitable candidate antigens for use as subunit vaccines;
(vi) Determine the role of regulatory T cells in the immunopathogenesis of mycobacterial infections in animals. The working hypothesis is that dysregulation of the immune response to M. paratuberculosis and M. bovis is, at least in part, attributable to development of regulatory T cells (Tregs). Evidence suggests that Tregs may be responsible for down-regulating effector memory CD4 cells in an antigen-specific manner. This hypothesis will be tested by characterizing cell surface markers of Tregs using flow-cytometeric and expression analysis techniques.