Progress 03/15/24 to 03/14/25
Outputs
Target Audience:Large animal veterinarians Dairy farmers Veterinary research community Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Our project offers training opportunities for many students, including: • one research technician with a training background in cattle health and nutrition • one DVM postdoc • one MS student, three PhD students, and one DVM-PhD dual degree student One PhD student in particular was selected to present our work at CRWAD meeting 2025. How have the results been disseminated to communities of interest?We have givenoral presentations on the project, including new and unpublished data, at CRWAD meetings 2024 and 2025. What do you plan to do during the next reporting period to accomplish the goals?1. Determine the genetic determinants of Lm pathogenesis in the bovine GI tract We plan to perform a transposon sequencing (TnSeq), a genome-wide approach to systematically identify genes required for Lm to colonize the bovine GI tract. 2. Complete profiling of mucosal immune responses We plan to complete histopathology assessments of infected tissues from other animals. We also plan to perform RNAseq of infected tissue to determine bovine host transcriptional response to Lm infection.
Impacts
What was accomplished under these goals?
1. Determine replication and invasion kinetics ofListeria monocytogenes (Lm) in the bovine intestines: A significant challenge of studying bovine listeriosis is the lack of a bovine infection model for Lm. We established and optimized a calf ligated ileal loop infection model for Lm.In this procedure, young calves are put under generalanesthesia and abdominal surgery was performed to ligate the ileum into ~38 loops. Each loop is injected with Lm or PBS (as mock infection). Ileal loops are harvested at 5 hpi and 8 hpi to determine Listeria burdens in intestinal lumen and tissue. Results: At an inoculum dose of 5x107 cfu per loop, Lm strain 10403S consistently infected the lumen and intestinal tissue in all of the six animals in our study. Luminal burdens were rather variable, with medians of ~103 cfu/g at 5 hpi and 106 cfu/g at 8 hpi. Intestinal tissue burdens were rather consistent, with medians of 102 cfu/g at 5 hpi and 5 x 103 cfu/g at 8 hpi. Increasing inoculum dose to 108 cfu per loop did not increase Lm burdens. 2. Determine the roles of virulence factors that had been studied in the mouse model of infection Very little is known about how Lm colonizes the bovine GI tract, including what genes mediate colonization. In the mouse model of infection, actA and hly genes are critical for Lm virulence. Furthermore, the general stress response factor Sigma B and the multidrug efflux pump repressor, TetR, have been shown to be important for survival under stress conditions, including conditions relevant to the gut such as osmotic and acid stresses. However, the roles of these genes in bovine infection are not known. We infected ligated ileal loops of three animals with the WT, delta-actA, delta-hly, delta-sigB, and delta-tetR strains, at the inoculum dose of5x107cfu per loop. Surprisingly, none of these mutants exhibited any infection defect, either in the intestinal lumen or in the tissue, compared to the WT strain. This result suggests that Lm pathogenesis in the bovine GI tract is complex and mediated by novel virulence factors that are not revealed in the mouse model. 3. Determine mucosal immunity to Lm infection We performed histopathology of the following tissue samples: un-infected, mock infected, and infected with wild-type Lm. At 8 hpi, there was no sign of inflammation. Please note that all these tissue samples came from one animal.
Publications
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Progress 03/15/23 to 03/14/24
Outputs
Target Audience:Large animal veterinarians Dairy farmers Veterinary research community Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Our project offers training opportunities for many students, including: one research technician with training background in cattle health and nutrition one DVM postdoc two MS students, twoPhD students, and one DVM-PhD dual degree student These trainees contributed to the oral presentation at CRWAD 2024 How have the results been disseminated to communities of interest?We have given an oral presentation on the project, including new and unpublished data, at CRWAD meeting 2024. The presentation was entitled "Colonization kinetics and mechanisms of Listeria monocytogenes infection in the bovine gastrointestinal tract". What do you plan to do during the next reporting period to accomplish the goals?1. We will examine the kinetics of luminal replication and intestinal invasion by different Listeria monocytogenes strains, including clinically relevant strains in the bovine host 2. We will examine the role of Listeria virulence factors in intestinal infection 3. We will determine the bovine mucosal immune responses to Listeria infection
Impacts
What was accomplished under these goals?
1. Optimization of bovine infection model for Listeria monocytogenes: A significant challenge of studying bovine listeriosis is the lack of a bovine infection model for Listeria monocytogenes. In the last period, we established and optimized a calf ligated ileal loop infection model. Briefly, young calves are put under general anesthesia and abdominal surgery was performed to ligate the ileum into ~38 loops. We have now optimized the experimental procedure including infectious dose and infection time. 2. Replication and invasion kinetics of Listeria monocytogenes in the bovine intestines Using Listeria monocytogenes strain 10403S, we found that Listeria can quickly establish colonization in the bovine intestines. We also observe replication of Listeria, by approximately 2-3 logs of Listeria cells between 5 and 8 hours post infection, to a high-density population in the intestinal lumen. A smaller fraction of Listeria is able to invade and replicate in the intestinal tissue.
Publications
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