Location: Infectious Bacterial Diseases Research
2023 Annual Report
Objectives
Objective 1: Through genomic–scale approaches, identify MAP antigens for improved detection of Johne’s disease in cattle, including early stage infection. Building on this approach, identify and test selected antigens that could be used as vaccine candidates.
Sub-objective 1.A: Design a synthetic antigen comprising strong epitopes from MAP antigens detected early in MAP infection of cattle.
Sub-objective 1.B: Use genomic approaches to discover new vaccine and diagnostic targets in MAP and improve on current diagnostic tests.
Sub-objective 1.C: Establish a CRISPR/Cas9 gene editing system in MAP for fast and targeted gene disruption to create live attenuated vaccine strains.
Objective 2: Characterize the host’s innate-immune response to onset of MAP infection in order to identify and develop novel intervention strategies to control infection with MAP.
Sub-objective 2.A: Characterize patterns of innate immune responses to natural infection in cattle in asymptomatic and clinical stages.
Sub-objective 2.B: Characterize the presence of innate immune cells in tissues at the site of infection from naturally infected cattle.
Approach
First identified over a century ago, Johne’s disease (a.k.a. Paratuberculosis) has long been recognized as a serious economic and animal health problem throughout the world in domesticated ruminants such as dairy and beef cattle, sheep, and goats. Paratuberculosis results in more than $200 million in annual losses to the U.S. dairy industry each year with additional losses incurred by the other species. The agent that causes this disease is the slow growing bacterium Mycobacterium avium subspecies paratuberculosis (MAP). A growing recognition of MAP infection in wildlife species is also of considerable concern, as is the recent implication of the presence of MAP in retail milk sources that may make this pathogen a risk from a milk quality and food safety standpoint. The overall goal of the work described in this project is to reduce the impact of Johne’s disease on the livestock producer and their corresponding industries. This goal is best accomplished by understanding the bovine immune response and what role specific immune cells play in order to inform next generation vaccine development and to identify antigens and assay platforms with the ability to detect the disease earlier, before transmission and disease spread occurs. With this goal and approaches in mind, we propose two primary objectives: (1) Through genomic–scale approaches, identify MAP antigens for improved detection of Johne’s disease in cattle, including early stage infection. Building on this approach, identify and test selected antigens that could be used as vaccine candidates; (2) Characterize the host’s innate-immune response to onset of MAP infection in order to identify and develop novel intervention strategies to control infection with MAP. This research will not only expand the basic knowledge of the disease, but it will lead to countermeasures to combat the disease, providing significant economic benefits to livestock producers.
Progress Report
In support of Objective 1, research continued in 1.A by testing synthetic peptides using well-characterized serum samples. Although we identified strongly reacting peptides through this approach, we found high background while screening synthetic proteins as they strongly reacted to the negative control samples. We utilized the contingency plan of obtaining peptides from a different manufacturer and used more stringent wash methods to reduce background. For Objective 1.B, our bioinformatics approaches to design primers by considering all copies of IS900 in the Mycobacterium avium subspecies paratuberculosis (MAP) genome allowed development of polymerase chain reaction assays with increased efficiency and sensitivity for detecting the pathogen. Research on the lipid antigens of Mycobacterium avium subspecies paratuberculosis identified a third unique lipid in MAP that complements two novel lipids we identified previously. A patent application is being submitted for this third novel lipid as a diagnostic antigen. In work addressing Objective 2.A, we overcome technical issues to obtain the yield of neutrophils needed to characterize innate immunity in MAP-infected cattle. Lastly, we determined which virulent MAP strains would be used in M1 and M2 macrophage experiments under Objective 2B.
Accomplishments
1. New, improved primers for detection of Johne’s disease in nucleic acid-based diagnostic tests. Mycobacterium avium subspecies avium (MAP) is a chronic debilitating disease that causes high economic costs for the dairy industry. ARS scientists at Ames, Iowa, used applied genomic data studies to improve performance of DNA-based PCR diagnostic tests. A IS900 insertion repetitive sequence, termed IS900, is present in the MAP genome at least 16 times and has been previously used as a target for polymerase chain reaction (PCR) assays. However, current PCR primers were designed using sequence data from a single copy of IS900 instead of considering all 16 copies collectively. The current project designed new primers based on all copies of IS900 collectively. Legacy and newly designed primers were compared in real time PCR assays. The new primers demonstrated improved sensitivity for detecting MAP in purified DNA and field samples. This research improved the sensitivity of PCR tests for detecting MAP infection in cattle. These findings will be of interest to animal producers, veterinarians, researchers, and diagnostic laboratories.
