Performing Department
(N/A)
Non Technical Summary
Liver abscesses affect 0 to 95% of feedlot cattle on an individual pen basis and in severe cases can decrease feed intake, average daily gain, gain:feed, and hot carcass weight of finishing cattle. The cost of liver abscesses, which are responsible for 70% of all liver condemnations in the United States is greater than $900 million annually because of condemned livers, increased carcass trimming, and diminished offal and carcass values. This financial burden to the cattle feeder and beef processor represents a decrease in the efficiency and sustainability of beef production.Results of the proposed studieswill be used to improve the quality and efficiency of producing beef and understanding metabolic disorders in beef cattle. These outcomes can be used to increase agriculture production to meet increased global food supply needs. For rapid dissemination, data will be submitted to regional, national, and international scientific and stakeholder meetings for presentation and high-impact learning experiences for graduate students.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
0%
Goals / Objectives
Objective 1: Evaluate barrier dysfunction and delineate bacterial pathogens causally associated with liver abscess formation using a minimally invasive model that mimics natural infection. Our working hypotheses are two-fold: 1) although liver abscesses are polymicrobial infections, F. necrophorum alone can initiate infection and T. pyogenes and S. enterica possibly contribute to infection but do not initiate abscess development; and 2) induction of barrier dysfunction (leaky gut) through ruminal acidosis, which mimics natural infection or indomethacin-induced enteropathy model can be used to change the integrity of the intestinal epithelium and induce leaky gut to facilitate bacterial translocation.Objective 2: Elucidate how Fusobacterium necrophroum adheres and modifies the transcriptional responses of epithelial cells derived from distinct areas of the gastrointestinal tract in beef cattle. Our working hypothesis is that F. necrophroum adheres to ruminal and hindgut epithelial cells and triggers pathological changes in the cellular inflammatory and metabolic gene profile. Revealing molecular mechanisms in which F. necrophroum is involved in the pathogenesis of liver abscesses is a key step towards the development of preventive strategies to mitigate the incidence of this disease in beef cattle.Objective 3: Characterize the effects of level and source of roughage NDF on the prevalence of liver abscesses in finishing beef cattle. Our working hypothesis is that an optimal source and dietary concentration of roughage NDF can be identified, which will be a practical and useful management strategy to decrease liver abscesses in the beef cattle industry.
Project Methods
Experiment 1. Forty weaned steer calves from a commercial calf ranch or dairy operation in the Texas Panhandle will be procured (initial body weight = 90 to 150 ± 25 kg) and used in a model study at the USDA Livestock Issues Research Unit near Lubbock, TX. Calves will be randomly assigned to 1 of 4 treatments (Figure 4): 1) high-starch acidotic diet and intraruminal inoculation of F. necrophorum subsp. necrophorum, S. enterica serovar Lubbock, and T. pyogenes (n = 10); 2) high-starch acidotic diet and intraruminal inoculation of F. necrophroum subsp. necrophorum(n = 10); 3) high-starch acidotic diet intraruminal inoculation of S. enterica serotype Lubbock (n = 10); or 4) high-starch acidotic diet and intraruminal inoculation of T. pyogenes (n = 10).For preparation of the inoculum, a pure culture of a high leukotoxin-producing strain of F. necrophorum, previously isolated from a liver abscess (strain FN-8LI) will be grown into 100 mL of pre-reduced, anaerobically sterilized brain heart infusion broth and incubated for 14 h at 37?. T. pyogenes and S. Lubbock will be cultured in 100 mL of Mueller Hinton broth for 24 and 12 h, respectively, under aerobic conditions at 37? without agitation. The bacterial concentration (CFU/mL) in the inoculum will be determined by serially diluting the culture and spread plating on blood agar plates.Experiment 1. Forty weaned steer calves from a commercial calf ranch or dairy operation in the Texas Panhandle will be procured (initial body weight = 90 to 150 ± 25 kg) and used in a model study at the USDA Livestock Issues Research Unit near Lubbock, TX. Calves will be randomly assigned to 1 of 4 treatments (Figure 4): 1) high-starch acidotic diet and intraruminal inoculation of F. necrophorum subsp. necrophorum, S. enterica serovar Lubbock, and T. pyogenes (n = 10); 2) high-starch acidotic diet and intraruminal inoculation of F. necrophorum spp. necrophorum (n = 10); 3) high-starch acidotic diet intraruminal inoculation of S. enterica serotype Lubbock (n = 10); or 4) high-starch acidotic diet and intraruminal inoculation of T. pyogenes (n = 10).Inoculation. Following the diet change cycles (day 21; Figure 5), the calves will be intraruminally administered the bacterial inoculation. A frick speculum will be placed in the oral cavity to the level of the oropharynx and flexible tubing will be inserted into the speculum, directed to the esophagus, and passed to the rumen. The bacterial inoculum will be poured into the flexible tubing through a funnel and the tubing will be flushed with 100 mL of sterile phosphate buffered saline.Calves will be fed the acidotic diet from day 22 to harvest on day 41. At harvest, calves will be evaluated for liver abscesses and/or scarring along with an evaluation of the rumen and colon. Ruminal and colonic tissues from calves will be cultured for F. necrophorum, T. pyogenes, and S. enterica. In addition, ruminal tissue, ruminal contents, colonic tissue, and colonic contents will be collected at harvest, and ruminal and colonic tissues will be cultured for F. necrophorum, T. pyogenes, and S. enterica. The abundance of F. necrophorum subsp. necrophorum in tissues and contents will be determined using quantitative PCR assay.Bacterial Culture of Liver Abscesses. When a LA is detected, the piece of liver with an intact abscess will be sliced, placed in a plastic bag on ice, and shipped overnight to Kansas State University for culture analysis to determine the prevalence and isolate F. necrophorum, T. pyogenes, and S. enterica. The procedures to isolate and identify the three species are described by Amachawadi et al. (2017). If samples do not yield Salmonella by direct plating, an enriched sample will be plated onto Hektoen Enteric agar for isolation (Amachawadi et al., 2017).Experiment 2. Forty weaned dairy steer calves from a commercial calf ranch or dairy operation in the Texas Panhandle will be used. Calves will be randomly assigned to 1 of 4 treatments: 1) control (n = 10) calves fed a low-starch control diet; 2) low-starch control diet with intraruminal inoculation with Fusobacterium necrophorum and Salmonella enterica spp. Lubbock with injections of indomethacin (used to induce leaky gut, described later) 48 hours before inoculation (n = 10); 3) acidotic diet with intraruminal inoculation with F. necrophorum and S. enterica serotype Lubbock (n = 10); or 4) acidotic diet with intraruminal inoculation with F. necrophorum and S. enterica serotype Lubbock with injections of indomethacin 48 hours before inoculation (n = 10). All methods besides the treatments will be identical to those in Experiment 1.Cell Adhesion Assay Design and Assessment (Figure.4). Primary BECs will be cultured in 6-well plates in DMEM/2% FBS until 80% confluency. To assess the F. necrophorum adhesion to BECs, a 100:1 multiplicity of infection of F. necrophorum colonies will be incubated with BECs in DMEM without fetal calf serum for 1 h at 37°C without oxygen, as described in the preliminary data section . For each condition, two wells of 6-well plates will not be stained with Wright-Giemsa and will be used for RNA collection and transcriptome analysis, as described below.Cell Adhesion Quantification. Cell adhesion will be quantified with a BioTek Cytation5 with Gen5 software.RNA Sequencing Analysis of Epithelial Cells. Isolated RNA will be submitted to the TTU Biotechnology and Genomics Laboratory for RNA quality assessment, library preparation and sequencing. Differentially expressed genes between control and F. necrophorum-exposed epithelial cells from different locations will be identified based on a robust Benjamini-Hochberg corrected false discovery rate (FDR) with a P-value < 0.05 (JMP 14 Pro).Experiment 1. Two hundred forty steers will be procured from auction markets located in the Southern Great Plains of the United States and fed until harvest. Experiment 1 will be a dose titration to determine the optimal level of roughage NDF in beef cattle finishing diets (4 treatments × 15 body weight blocks × 4 steers/pen). The levels of roughage NDF will be 3, 4, 5, and 6% (Figure 7). These levels encompass the roughage NDF levels currently fed in the feedlot industry (Samuelson et al., 2016). The roughage source will be corn stalks, which is a commonly used roughage in the feedlot industry. Body weights will be measured every 28 days. Ruminal fluid will be collected via stomach tube at the beginning and approximate mid-point of days on feed and before harvest to determine abundance of F. necrophorum based on q-PCR assay.Experiment 2. A 2 × 2 factorial arrangement of treatments will be used in a randomized complete block design with 240 steers (4 treatments × 15 body weight blocks × 4 steers/pen). Factors will consist of roughage source (corn stalks or corn silage) and the two roughage NDF levels from Exp. 1 that resulted in the least percentage of liver abscesses. Thus, the resulting 4 treatment combinations will consist of 1) corn stalks with the first level of roughage NDF determined in Exp. 1 that incurred the least LA; 2) corn stalks with the second level of roughage NDF determined from Exp. 1 that incurred the second least percentage of LA; 3) corn silage with the first level of roughage NDF determined in Exp. 1 that incurred the least LA; and 4) corn silage with the second level of roughage NDF determined from Exp. 1 that incurred the second least percentage of LA (Fig. 8). Body weights will be measured every 28 days. Ruminal fluid will be collected as described in Exp. 1 and used to determine the abundance of F. necrophorum by q-PCR.