Progress 02/01/23 to 01/31/24
Outputs
Target Audience:The target audience for this project is catfish producers in the commercial sector. The goal of this study is to identify potential intervention strategies to reduce catfish mortality associated with bacterial infections. Another target audience is veterinarians, fish disease researchers, microbiologists, diagnostic and extension specialists seeking new knowledge and preventive methods to reduce prevalence and impact of disease caused by Edwardsiella ictaluri. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Two graduate students and one research scientist have been trained on this project. How have the results been disseminated to communities of interest?The results from this project were presented at a Fish Health Update seminar for East Mississippi catfish growers. The seminar was facilitated by Mississippi State University Extension Service, and its purpose was to provide an update on current activities, research findings, and treatment recommendations for major fish health issues. What do you plan to do during the next reporting period to accomplish the goals? Comparative genomic analysis will be conducted to identify specific point mutations and to understand the molecular basis for attenuation in D30-adapted and D60-adapted strains. We will assess the impact of TC treatment on ESC severity by determining pathological changes and quantifying E. ictaluri in catfish tissues. We will evaluate the effect of dietary-TC on intestinal immune response of catfish following E. ictaluri infection.
Impacts
What was accomplished under these goals?
Determine the major mechanism(s) of TC antibacterial action. To elucidate the antibacterial mechanism of TC, quantitative proteomics was performed to investigate differentially expressed proteins (DEPs) in E. ictaluri in response to TC treatment. The minimum inhibitory concentration (MIC) of TC against E. ictaluri was determined by microdilution method.E. ictaluri were cultured into media containing 2/4 × MIC and 3/4 × MIC of TC in four biological replicates and incubated at 30 °C for 18 hours. The bacterial pellets were collected, washed, lysed, and used for peptide fractionation and data-independent acquisition (DIA). Bioinformatics analysis suggested efflux pump, a membrane fusion protein, and efflux transmembrane transporter are upregulated in sub-MICs of TC. Potential of E. ictaluri to develop resistance to TC. The goal of this experiment was to determine the influence of successive treatments with TC on E. ictaluri. Briefly, E. ictaluri 93-146 strain was subjected to serial passage in sub-MICs of TC (0.016 µl/ml). The process was repeated every 24 hours for 60 consecutive days. D30-adapted (30dayspassage in sub-MICs of TC) and D60-adapted (60 days passage in sub-MICs of TC) strains were selected and stored at -80 °C for analysis. The disc diffusion and broth dilution methods were used to compare the inhibition zone and MIC of adapted strains (D30-adapted and D60-adapted) and non-adapted E. ictaluri 93-146 (control) against TC and antimicrobial agents, including florfenicol and tetracycline. Overall, the disk diffusion assay and MIC revealed no significant differences in the inhibition zones and MIC values between adapted and non-adapted E. ictaluri, for TC itself, tetracycline, and florfenicol. Additionally, we analyzed the proteomic alterations in the D30-adapted and D60-adapted strains compared to E. ictaluri 93-146. Based on enrichment analysis, D30-adapted and D60-adapted strains showed significant upregulation in the pyrimidine and carbon metabolism and metal-ion binding function. In addition, we observed a significant downregulation of type III and type VI secretion systems in D30-adapted and D60-adapted strains compared to E. ictaluri 93-146. Type III and VI secretion systems play crucial roles in delivering virulence factors to host cells. It is possible that disruption of these two systems impacts virulence of E. ictaluri 93-146. The pathogenicity of D30-adapted and D60-adapted strains was compared to control E. ictaluri 93-146 strain in catfish fingerlings using immersion challenge protocol. Following a 21-day post-infection period, 88% mortality was observed in catfish infected with E. ictaluri 93-146, while no mortality was recorded for D30-adapted and D60-adapted strains. Significant results achieved. TC had a significant antimicrobial effect on E. ictaluri. The fish experiments showed that incorporation of TC in catfish feed significantly lowered mortality associated with E. ictaluri infection compared to fish fed the control diet. TC has the potential to mitigate ESC-related losses. D30-adapted and D60-adapted strains were generated after serial passage of E. ictaluri 93-146 on culture medium containingTC. There was no substantial change in the MIC of D30-adapted and D60-adapted strains compared to the control. D30-adapted and D60-adapted strains lost virulence. Key outcomes or other accomplishments realized. A disc diffusion assay revealed that TC possesses potent antimicrobial properties against E. ictaluri. Results from challenge trials indicated that supplementation of catfish diets with TC increases catfish survival rates from E. ictaluri infection. The catfish group fed a diet containing TC and cod liver oil had significantly lower mortality compared to fish received a regular diet following E. ictaluri infection. The development of resistance to TC and cross-resistance to other antimicrobial agents was not observed after exposure of E. ictaluri to TC for 60 passages. E. ictaluri did not develop resistance to TC or other antimicrobial agents due to continuous and prolonged exposure to TC.
Publications
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2023
Citation:
Rostami S, Riman MM, Abdelhamed H. Effect of dietary Trans-cinnamaldehyde on susceptibility of catfish to Edwardsiella ictaluri. Conference of Research Workers in Animal Diseases- January 20-24, 2023, Chicago, IL. Oral presentation.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2023
Citation:
Rostami S, Riman MM, Abdelhamed H. Effects of dietary Trans-cinnamaldehyde on health status of channel catfish challenged with Edwardsiella ictaluri. 87th Annual Mississippi Academy of Sciences Meeting February 23-24, 2023 Biloxi Convention Center Biloxi, MS. Oral presentation.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2024
Citation:
Rostami S, Abdelhamed H. Investigate the impact of long-term exposure of Edwardsiella ictaluri to trans-cinnamaldehyde on resistance development. Conference of Research Workers in Animal Diseases January 20-24, 2024, Chicago, IL.
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Progress 02/01/22 to 01/31/23
Outputs
Target Audience:The target audience for this project is catfish producers in the commercial sector. The goal of this study is to identify potential intervention strategies to reduce catfish mortality associated with bacterial infections. Another target audience is veterinarians, fish disease researchers, microbiologists, diagnostic and extension specialists seeking new knowledge and preventive methods to reduce prevalence and impact of disease caused by Edwardsiella ictaluri. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Two graduate students and one research scientist have been trained on this project. How have the results been disseminated to communities of interest?The results from this project were presented at a Fish Health Update seminar for East Mississippi catfish growers in Macon, Mississippi, on June 29, 2022. The seminar was facilitated by Mississippi State University Extension Service, and its purpose was to provide an update on current activities, research findings, and treatment recommendations for major fish health issues. What do you plan to do during the next reporting period to accomplish the goals?We will assess the impact of TC treatment on ESC severity by determining pathological changes and quantifying E. ictaluri in catfish tissues. We will evaluate the effect of dietary-TC on intestinal immune response of catfish following E. ictaluri infection.
Impacts
What was accomplished under these goals?
Aim 1. Determine efficacy of dietary-TC to prevent and treat E. ictaluri infection in catfish. The goal of this experiment was to assess the ability of various concentrations of TC to protect catfish fingerlings against E. ictaluri infection. Specific pathogen free (SPF) catfish were stocked into 40 L tanks at a rate of 10 fish per tank. The tanks were divided into seven groups of five replicates. Two control groups were included: fish fed florfenicol-medicated diet and another group fed the regular, basal diet. For optimizing the TC concentration, TC was incorporated into commercial feed at five different concentrations (10, 15, 25, 30, and 35 ml/kg). Challenges were conducted by immersion of fish in water containing 107 CFU/ml of E. ictaluri strain 93-146, and percent mortalities following challenge was evaluated. TC-diet at 15 ml/kg provided maximum protection against E. ictaluri compared to control group (35.86% vs 82.22%). This suggests the dietary-TC has the potential to limit the ESC-related losses. A challenge trial was conducted to evaluate the ability of dietary TC and cod liver oil (commonly used by catfish industry) to protect catfish fingerlings against E. ictaluri infection. SPF catfish fingerlings were stocked into thirty tanks (10 fish/tank). Fish were divided into six groups of five replicate tanks. The first group was fed a regular diet (as a negative control group). The second group was fed TC at a concentration of 15 ml/kg feed plus cod liver oil 5 ml/kg. The third group was fed TC at concentration of 15 ml/kg feed plus 10 ml/kg cod liver oil. The fourth group was fed TC at concentration of 15 ml/kg feed plus 15 ml/kg cod liver oil. The fifth group was fed TC at concentration of 15 ml/kg feed plus 25 ml/kg cod liver oil. The last group was fed TC at concentration of 15 ml/kg feed plus 35 ml/kg cod liver oil. The mortality in catfish fed the TC-diet at 15 ml/kg plus cod liver oil at 5 and 25 ml/kg (25.25% and 24.24%) was significantly lower compared to those fish received regular diet (71.33%). Aim 2. Explore the mechanisms by which TC-supplemented diet provides protection of catfish against E. ictaluri. Determine the killing efficiency and phagocytosis of macrophages. The goal of this experiment was to compare the killing activity of macrophages in TC fed catfish vs catfish fed basal diet. Catfish were divided into three groups of five replicate tanks. Group 1 received a basal diet without supplementation and infection. Group 2 received a basal diet and was infected with E. ictaluri. Group 3 received diet supplemented with TC (15 ml/kg feed) and cod liver oil (5 ml/kg feed). Fish were challenged by immersion with green fluorescent protein (GFP)-labeled E. ictaluri strain 93-146 (107 CFU/ml of water). Ten fish (2 fish/tank) were euthanized from each group at 1, 3, 5, 7, 10, and 14 days after TC application. Anterior kidney (AK) was dissected from each fish and used for collection of AK derived macrophages using gradient centrifugation with Histopaque 1077. Flow cytometry was applied to assess the rate of phagocytosis of E. ictaluri by macrophages in TC fed catfish vs catfish fed basal diet (data analysis in progress). Aim 3. Evaluate the ability of E. ictaluri to develop resistance to TC and determine the major mechanism(s) of TC antibacterial action. Study of the tendency of E. ictaluri to develop resistance to TC. The goal of this experiment was to determine the influence of a continuous treatment with TC on E. ictaluri. In this study, E. ictaluri 93-146 strain was subjected to serial passage in subinhibitory concentrations of TC. The process was repeated every 24 hours for 60 consecutive days. After passage number 30 and passage number 60, frozen stock was prepared and stored at -80 C. The disc diffusion and broth dilution method were used to compare the inhibition zone and MIC of treated (passage 30 and 60) and non-treated E. ictaluri against TC and antimicrobial agents, including florfenicol and tetracycline. Specific objectives met -Aim 1. In vivo challenge experiments were performed to determine the optimal dietary trans-cinnamaldehyde (TC) feeding regime in catfish fingerlings. Catfish were infected with E. ictaluri via immersion and received either TC-supplemented diet or regular feed. Dead fish were recorded every day and used to determine percent mortality. In addition, we evaluated the ability of basal diet contains TC and cod liver oil to protect catfish fingerlings against E. ictaluri infection. Results indicated that catfish fingerlings fed with dietary supplemented with TC and cod liver oil showed enhanced survival rate compared to the control. -Aim 2. We investigated the phagocytosis of macrophages in the catfish received either regular diet or TC-supplemented diet after immersion challenge with GFP-labeled E. ictaluri. Macrophages were harvested from catfish at different time points following infection. Flow cytometry was used to assess the phagocytosis rate of E. ictaluri by macrophages in TC fed catfish in comparison to catfish fed basal diet. -Aim 3. We investigated the ability of E. ictaluri to develop resistance to TC after continuous TC exposure (30 passage and 60 passages). We also investigated cross-resistances of E. ictaluri to different antimicrobial agents after continuous TC exposure. After the TC exposure, the disk diffusion assay and MIC revealed no significant differences in the inhibition zones and MIC values between treated and non-treated E. ictaluri, in particular for TC itself, tetracycline, florfenicol. Significant results achieved. TC had a significant antimicrobial effect onE. ictaluri. The fish experiments showed that incorporation of TC in catfish feed significantly lowered mortality associated with E. ictaluri infection compared to fish fed the control diet. TC has the potential to mitigate ESC-related losses. There was no substantial change in MIC, as measured using zones of inhibition, of E. ictaluri after serial passage with TC. Key outcomes or other accomplishments realized. A disc diffusion assay revealed that TC possesses potent antimicrobial properties against various pathogens, includingE. ictaluri, Flavobacterium columnare, and Aeromonas hydrophila ML09-119. Result from challenge trials indicated that supplementation of catfish diets with TC increases catfish survival rate after E. ictaluri infection. The catfish group fed diet containing TC and cod liver oil had significantly lower mortality compared to fish received a regular diet following E. ictaluri infection. The development of resistance to TC itself and cross-resistance to other antimicrobial agents was not observed after exposure of E. ictaluri to TC for 60 passages. E. ictaluri did not develop resistance to TC itself or other antimicrobial agents due to continuous and prolonged exposure to TC.
Publications
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2022
Citation:
Abdelhamed H. Effect of dietary Trans-cinnamaldehyde on susceptibility of catfish to Edwardsiella ictaluri. Conference of Research Workers in Animal Diseases (CRAWD. 2022), Chicago, IL. (Oral presentation).
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2023
Citation:
Sahar Rostami, Munshi Mustafiz Riman, Hossam Abdelhamed. Effects of dietary Trans-cinnamaldehyde on health status of channel catfish challenged with Edwardsiella ictaluri. 87th Annual Mississippi Academy of Sciences Meeting February 23-24, 2023 Biloxi Convention Center Biloxi, MS. (Oral presentation).
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2022
Citation:
Hanson L and Abdelhamed H. Examples of Global, National, and Regional AMR and Biosecurity Activities at Mississippi State University. FAO Reference Center meeting on AMR and Aquaculture Biosecurity. Virtual meeting. November 20, 2022. (Oral presentation).
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2022
Citation:
Hanson L and Abdelhamed H. Sustainable research program on AMR. Fish Health Seminar in Macon, MS on June 29, 2022 (Oral presentation).
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