Role of host & bacterial factors in persistence of Salmonella in the bovine lymphatic system

Salmonella is a bacterium that causes severe diarrhoea in humans. Infections are often acquired via the food chain and environment from farmed animals owing to the ability of Salmonella to persist in their bodies. Around 94 million human cases and 155 thousand deaths occur worldwide each year. Cattle are a significant source of such infections, partly because Salmonella can leave the bovine gut via a branching network of vessels and nodular glands termed the lymphatic system. The lymphatic system normally helps to fight infection, however some types of Salmonella are adapted to survive in lymph nodes and evade the immune system. Removal of such lymph nodes is not possible on the scale of modern beef production as they are small, widely dispersed and hard to access. As a consequence, they are often incorporated into ground beef for human consumption. Large outbreaks due to ground beef have occurred, including by strains resistant to frontline medicines, and a need exists to devise strategies to prevent or reduce Salmonella infection in cattle. Such strategies are also required to enhance animal health as Salmonella are a significant cause of diarrhoea, blood-poisoning and abortion in cattle.

The Stevens laboratory has been at the forefront of research to understand how Salmonella colonises the intestines of cattle and, in some cases, migrates around the body via the lymphatic system. We have developed a novel surgical model in which we insert tubes into the blood and lymphatic systems to capture bacteria as they migrate from the gut. This has shown that escape from lymph nodes via the lymphatic system can partly explain why some strains spread around the body, whereas others are confined to the gut or cleared. A key gap in our knowledge is how Salmonella enters the lymphatic system in the first place. Almost nothing is known about the host cell types with which Salmonella associates in the bovine gut or whether these migrate to the lymphatic system. The consequences of such interactions for Salmonella and the development of protective immune responses are unknown. Such gaps in knowledge are constraining our ability to design strategies to control infection. The Hopkins and Hope laboratories conduct world-leading research to understand the nature and consequences of interactions between infectious agents and cells of the ruminant immune system, which in turn is helping to improve vaccines for tuberculosis, Johne's disease, viruses and parasitic infections. Our proposal brings together substantial cash investment from Pfizer Animal Health and researchers with complementary expertise to:

1. Understand how Salmonella enters the lymphatic system of cattle. This will involve inserting tubes into lymph vessels draining to lymph nodes at various sites in the gut of young calves and older steers. We will examine which cell types interact with Salmonella, how they respond and what happens to the bacteria. We will then ask whether such events can explain why some types of Salmonella are cleared by cattle whereas others spread around the body. The information will help us to target vaccines to relevant cell types and boost beneficial responses.

2. Determine if different cattle-associated strains of Salmonella are equally able to spread through the lymphatic system. This will tell us whether vaccines or drugs must target a wide range of Salmonella strains, or focus on high-risk types.

3. Identify Salmonella genes required for survival in the lymphatic system. We recently used a novel method to simultaneously survey the ability of hundreds of Salmonella mutants, each lacking a different character, to colonise the bovine gut wall. We are able to retrospectively apply this method to lymph nodes from the same animals to identify factors that influence persistence in the lymphatic system. Such factors may be suitable for inclusion into vaccines or as targets for drugs.

Persistence of Salmonella in the bovine lymphatic system leads to contamination of ground beef and is associated with the systemic virulence of host-restricted serovars. The mode of delivery of Salmonella from the intestinal mucosa to draining lymph nodes and the nature and consequences of interactions with host cells in this process are unknown. Moreover, it is unclear if cattle-associated serovars are equally able to enter the bovine lymphatic system and evade clearance. This proposal brings together complementary expertise and substantial investment from Pfizer Animal Health to define the role of host and bacterial factors in persistence of Salmonella in the bovine lymphatic system. Specifically, we will define the mode of translocation of S. Dublin to lymph nodes draining the intestines of calves and steers by cannulation of afferent lymph vessels. We will define the cell tropism of S. Dublin, fate of the intracellular bacteria and cellular responses, and determine how such events vary with serovar Typhimurium, Gallinarum and Newport strains toward an explanation of their differential virulence in cattle. We will also examine the spatio-temporal dynamics of spread of signature-tagged strains of cattle-associated serovars to define the extent to which interventions must confer cross-serovar protection. We also propose to identify Salmonella genes mediating persistence in the bovine lymphatic system by retrospective application of transposon-directed insertion-site sequencing to archived lymph nodes from cattle infected with 8550 random S. Typhimurium mutants. We have assigned phenotypes for >90% of such mutants in the distal ileal mucosa and quantitative assessment of their abundance in cognate nodes will identify niche-specific virulence factors and targets for intervention. The project extends our productive BBSRC-funded research using unique models in a species of strategic importance to food security and safety.

Cattle play an important role in global food security and bovine salmonellosis constrains both animal welfare and food safety. Contamination of ground beef is a significant concern, particularly in the United States where large multi-state outbreaks have occurred. Salmonella evolution has been punctuated by the emergence of epidemic and multi-drug resistant variants that have caused substantial harm in cattle and humans (e.g. S. Typhimurium definitive-types 104, 204 and 204c). The basis of the rise and decline of epidemic variants and reasons why some serovars cause acute diarrhoea while others cause sepsis and abortion are not understood. Prophylactic use of antibiotics is prohibited in many countries and therapeutic use is restricted owing to the potential for entry of residues into the food chain. Removal of contaminated lymphatic tissue is not feasible on the scale of modern beef production, where some abattoirs in the US process >5000 cattle a day. In this context, vaccines that prevent persistence of Salmonella in the bovine lymphatic system, or treatments that accelerate clearance, are highly desirable. The project will yield valuable data to underpin the design of such strategies, as reflected in the substantial investment of Pfizer Animal Health - a global leader in the development of veterinary vaccines and medicines. The project will also yield novel data on the repertoire and function of antigen-presenting cells serving the bovine intestines, and the mechanisms leading to protective innate and adaptive responses to enteric pathogens. Such data will benefit academia and industry in their efforts to control other ruminant intestinal diseases, including those caused by viruses and parasites. It may also identify immune signatures associated with infection or vaccination that can be exploited in diagnostic tests, as evidenced by an award-winning assay devised by Dr Hope to differentiate BCG-vaccinated cattle from those with tuberculosis. Identification of serovars and genes associated with persistence of Salmonella in the bovine lymphatic system will also yield markers for risk analysis and aid the targeting of control strategies.

Intellectual property arising from the project will be identified, protected and exploited as described in Pathways to Impact. Though we will primarily focus our impact activities on control of zoonosis and cattle health via links with Pfizer, the project will also benefit those studying other host-pathogen interactions and instil training in diverse areas. In particular, it will nurture skills in molecular microbiology, immunology, pathogenesis and surgery in animals of significance to food supply and safety. Training will extend beyond those deployed on the project to undergraduate, Masters and doctoral students via the research-led teaching of the applicants. Academics and Policy Makers will be informed of the nature and implications of the research via scientific and lay publications, presentations at symposia, invited lectures and via the comprehensive websites of the investigators. Moreover, the applicants actively engage with varied societies and forums to discuss issues affecting animal health and strategies for disease control. Materials and data arising from the project will be made available for legitimate uses on request, subject to publication and scrutiny for IP. Exchange of staff and students will promote knowledge transfer.

The project will also raise issues and data of importance to the public. The applicants have used short films, public speeches and lay articles to convey the purpose and importance of their work. This has included public engagement on food safety, ruminant diseases and methods to reduce, refine and replace animal use in research. The investigators will also explore ways to educate school children, for example by extending established links with the National Centre for Biotechnology Education and the STEM ambassador scheme.