Source: TEXAS STATE UNIVERSITY submitted to
DEVELOPMENT OF NASAL AND GUT PROBIOTICS TO PROTECT INTENSIVE CATFISH CULTURE AGAINST EDWARDSIELLA ICTALURI INFECTIONS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
OTHER GRANTS
Reporting Frequency
Annual
Accession No.
1026329
Grant No.
2021-70007-34786
Cumulative Award Amt.
$298,560.00
Proposal No.
2020-05418
Multistate No.
(N/A)
Project Start Date
Sep 1, 2021
Project End Date
Aug 31, 2024
Grant Year
2021
Program Code
[AQUA]- Aquaculture Research
Project Director
Huertas, M.
Recipient Organization
TEXAS STATE UNIVERSITY
601 UNIVERSITY DRIVE
SAN MARCOS,TX 78666
Performing Department
(N/A)
Non Technical Summary
The catfish industry losses millions of dollars every year due to bacterial disease in poor water quality systems. We propose that infection in fish is linked to the damage of beneficial microbiome in the nose and gut, by aquaculture water with high amounts of fish waste. Thus, recovering the microbiome of main external organs with probiotic treatments, can improve the fish immune system, and therefore reduce sickness.In this project, we will identify catfish nose and gut microbiome that are tolerant to poor quality waters. Then, we will design and produce probiotic treatments for nose and gut of fish and will assess their ability to protect against infections. Specifically, we will target for probiotic treatments that protect from the "hole in the head" disease in catfish, with our long-term goal being to develop food probiotics that can prevent bacterial illness in intensive culture systems.
Animal Health Component
100%
Research Effort Categories
Basic
(N/A)
Applied
100%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3113710104025%
3113710102025%
3113710110050%
Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3710 - Catfish;

Field Of Science
1040 - Molecular biology; 1100 - Bacteriology; 1020 - Physiology;
Goals / Objectives
Our long-term goal is to develop nasal probiotics that can protect catfish against enteric septicemia of catfish (ESC) in intensive culture systems with poor water quality. Thus, this proposal focuses on identifying nasal and gut microbes that can be altered by normal culture conditions and rendered sensitive to bacterial infection. The significance of this proposal is that it will describe the nasal and gut catfish microbiome and its dynamics under poor water quality conditions. Second, is that it will reveal a new screening technique for nasal and chronic asymptomatic bacterial infections, by using olfactogram and encephalogram to immediately assess bacterial detection and posterior infection without performing clinical trials. Third, we will design and test potential probiotic treatments that can establish future steps to create new sustainable treatments in aquaculture. This project will result in the identification of bacterial strains and catfish feed formulation that is intended to be used as nasal probiotic in aquaculture. A nasal probiotic in catfish is an environmentally sustainable treatment that can dramatically reduce the financial losses caused by ESC in catfish culture. The outcomes of our proposed experiments will be applicable to other aquaculture species sensitive to Edwardsiella such as bass or tilapia, other important aquaculture species in the US. Moreover, it will have direct translational applications in the fields of fish farming and animal health. For instance, developing probiotic treatments against IHNV, a rhabdovirus that accesses trout brain through the nose, could be achieved by exploiting the combination of olfaction screening techniques and identified probiotics. Additionally, understanding how nitrites can modulate the nasal microbiome can improve animal health by refining the requirements of nitrogenous waste in aquaculture.Our experiments will complete the following objectives:1. Identify changes in catfish nasal and gut microbiomes exposed to several sub-acute concentrations of nitrite. This objective will include the following experiments.- Exposure of catfish to chronic nitrite exposure and histological analysis (2 months, 0.6 effort during first year). We will use $6000 for the transport of fish, tagging supplies, fish maintenance (food, heaters, kits and sondas for measurement of water quality), and $4500 for glassware, miscellaneous chemical and supplies for histology. We will also acquire a computer and software to analyze data for the full project ($3000).- Isolation and identification of microbiome by whole genome shotgun (8 months, 0.95 effort during first year). We will use $47000 for shotgun metagenomic sequencing of 180 samples (DNA extraction kits, Nextera library preparation, 6Gb per sample NovaSeq Illumina (2x150bp); Isolation and identification of potential probiotic strains (microbiological media, PCR reagents, gel purification, Sanger sequencing reagents and consumables).2. Develop a screening method to evaluate olfactory infection of E. ictaluri or ESC vaccine in catfish exposed to nitrites. This objective will include the following experiments.-Electro-olfactogram recordings to measure olfactory response of catfish to food odorants and vaccine, prior and posterior to ESC infection (2 months, 0.35 effort during first year). We will use $5000 for electrophysiology consumables (electrodes, electrode holders, dissection material, and tubing) and $3000 for glassware, miscellaneous chemical and supplies.3. Identify and test potential nasal and gut probiotic strains to protect catfish against E. ictaluri in catfish reared with nitrites. All this experiments will be perform in the second year. This objective will include the following experiments.- Identification of bacterial strains isolated in aim 1showing high in vitro inhibitory activity to the pathogen E. ictaluri. (6 months, 0.8 effort during second year) We will use $6597 for Confirmation of probiotic colonization (DNA extraction kits, DNA quantification reagents, qPCR reagents, Primer designing and consumables),4 and $6500 for glassware, miscellaneous chemicals for culture.- Clinical experiments to pinpoint potential live probiotic that will protect catfish from E. ictaluri infection. (6 months, 1.1 effort during second year). We will use $6000 for the transport of fish, tagging supplies, fish maintenance (food, heaters, kits for measurement of water quality), 2000 for probiotic food production ,and $4000 for glassware, miscellaneous chemical and supplies.- We will also attend to aquaculture conferences and communicate with interested parts in catfish culture during the two-year project. We have $2000 allocated for travelling the first year and $3000 for the second. We also have $1000 allocated for publication and $1000 for renewal of software in the second year.
Project Methods
AIM #1: Identify changes in catfish nasal and gut microbiomes exposed to several sub-acute concentrations of nitrite.We will confirm the toxicological effect of nitrites in catfish nose and gut mucosa by measuring tissue damage and microbiome composition. To that end, catfish (3g body weight) will be acclimated in 35 L flow through aquaria with aerated water at room temperature (22°C) and without carbon filter or bio-balls for 7 days. Flow through aquaria will be controlled by using water release by gravity from an above 200 L plastic water tank. Fish will be fed daily commercial brand food pre and post nitrite exposure. After the acclimation period, fish will be exposed to nitrite concentrations 0 mM and 2.0 mM for 60 days. Each treatment will consist of three replicate tanks with 15 fish per replicate. Daily water samples will be taken for water quality assessment. At the end of the experiment, catfish olfactory epithelia and gut will be dissected for histological analysis and for whole genome shotgun (WGS) sequencing. Total metagenomic DNA from nasal epithelia and fecal material will be isolated. WGS metagenomic sequencing of the samples will be performed at the DNA Sequencing Facility at University of Wisconsin-Madison, and in house quality control will be used to further clean up the sequencing reads using fast toolkit (http://hannonlab.cshl.edu/fastx_toolkit/index.html). Next, sequencing adaptors, low-quality base calls, and low-quality sequencing reads will be removed from further analyses. Filtered reads will be taxonomically classified using the Kaiju software. Functional annotation to KEGG Orthologous will be done using the FMAP pipeline. We will use DESeq2 to identify differentially abundant KO genes or microbial species between the experimental groups. Additionally, reads will be assembled using MEGAHIT and metagenome-assembled genomes (MAGs) will be binned using MaxBin. Contamination of MAGs will be checked using CheckM. Functional annotation of MAGs will be performed using Prokka. One-way analysis of similarity (ANOSIM) and principal coordinates analysis (PCoA), using Morisita index, will be performed with PAST software version 3.152. The significance of the relative abundance of bacterial species difference will be performed using Kruskal-Wallis and multiple test correction via Benjamin and Hochberg false discovery rate, which are implemented in the STAMP software version 2.1.33.AIM #2: Evaluate olfactory detection and physiological responses to exposure of E. ictaluri or ESC vaccine in catfish exposed to nitrites. To evaluate olfactory sensitivity to pathogen in catfish exposed to nitrites, we will record the nasal responses to live attenuated E. ictaluri by electro-olfactogram. The olfactory potency will be determined using EOG recording, which measures the DC field electrical potential generated by olfactory neuronal receptors after activation by a chemical stimulus. Olfactory responses to stimuli will be DC electrical potential recorded by borosilicate microtubes filled with 3M KCl and connected to solid-state electronics, were electric signals will be amplified, filtered and digitized. The reference electrode and the recording electrode will be placed in the skin near the nose and between two posterior lamellae, respectively. Stock solutions of L-serine and E. ictaluri will be diluted and tested by increasing concentrations of odorants. Fish will be grouped by high olfactory responses and no olfactory responses to bacteria. Two days after exposure fish will be retested for olfactory responses, and clinical symptoms will be monitored for 4 weeks. Results will be analyzed with Prism software.AIM #3: Identify and test potential nasal probiotic strains. We will evaluate the in vitro and in vivo antagonistic activity of probiotic candidates against E. ictaluri. identified in Aim 1.Probiotic formulation. Bacterial isolates of E. ictaluri will be obtained from olfactory epithelia and the gut content of healthy catfish. Bacterial colonies exhibiting unique morphology will be isolated through sequential streaking and cultured. Cell pellets will be obtained and DNA will be extracted and quantified. The bacterial isolates will be identified by DNA sequencing of the 16S rRNA gene. The PCR products will be sequenced on the ABI 3500xL platform. Bacteria identification will be achieved by comparison of the 16S rRNA gene sequences against the Ribosomal Database Project (http://rdp.cme.msu.edu/) database, using the RDP classifier tool using an identity threshold of ≥ 80%. For determination of antimicrobial activity against E. ictaluri, pure cultures will be inoculated in agar plates containing one indicator strains against the pathogen. The diameter of inhibition zones will be measure after 24 and 48h.Clinical experiments. For this experiment, sexually mature catfish will be paired and allowed to naturally spawn in raceways at the San Marcos hatchery. A subsample of resulting larvae will be reared for 10 days to juvenile stage with two different probiotic treatments: control group (inactive bacteria in food) and treatment (with active bacteria). After 10 days of probiotic treatment, fish from each group will be transferred to 30 L aquarium. Then, fish will be nasally sprayed with 1 ml 106 CFU/mL and symptoms monitored for two weeks. For gut infections, 1ml of the diluted E. ictaluri suspension will be added to the aquarium water for a final dilution of 3 × 106 CFU/mL. The gut infection trial will last four days. In order to evaluate the protective activity of the probiotic under nitrite exposure, we will conduct these experiments under two nitrite conditions: no nitrite added and 1.0 mM nitrite addition. Therefore, each probiotic test (nasal and gut) will consists of four groups: (1) fish exposed to active bacteria with no nitrite; (2) fish exposed to active bacteria with 1.0 mM nitrite; (3) fish exposed to inactive bacteria in water with no nitrite; (4) fish exposed to inactive bacteria in water with 1.0 mM nitrite. Each group will have four 30 L aquarium replicates with 15 fish per tank. During the experiment, we will collect survival data and ESC symptoms, such as petechial hemorrhages, depigmented ulcers, abnormal behavior, eye bulging and abdominal fluid build-up and head ulcers. At the end of the experiment fish will be sampled for nose and gut tissue to confirm probiotic colonization. For the confirmation and quantification of probiotic colonization, specific primers targeting the 16S rRNA gene of the probiotic candidates will be designed using ARB software. Total bacterial abundance will be obtained using universal 16S rRNA gene primers.EFFORTS Additionally to our research activity, we will pursue the training and education of minority undergraduates and graduate students; stimulate the active engagement and promotion of women in STEM, attend scientific conferences to disseminate our results related to animal and human health and communicate with catfish farms in Texas and around the country.EVALUATIONThe project lead will be responsible for ensuring goals and deliverables are met according to our timeline. The results from this project will be disseminated in between TPWD aquaculture stations, aquaculture meetings, and publication of papers. The proposed project timeline and deliverables will be explicitly discussed with the USDA NIFA Aquaculture Program personnel by the project lead.TIMELINE Year 1: All animal work and sample collection will be performed in the first two months. After that, we will complete the experiments proposed in AIM #1 and AIM#2.Year 2 : We will perform all experiments propose in AIM #3 and write a manuscript derived from the award. We will also attend to aquaculture conferences and communicate with interested parts in catfish culture.

Progress 09/01/23 to 08/31/24

Outputs
Target Audience:We reached out to the catfish community during Aquaculture America 2024 (San Antonio, Texas) and presented a review of our work in a USDA grants session. In the Aquaculture America meeting, we interacted with both scientists and catfish farmers and shared information about our research. We are aiming to write a new project related with nitrites and catfish aquaculture with colleages in Mississipi. The PI gave two international seminal talks that allowed the interaction with aquaculturist in Europe and further the work of control of nitrogenous compounds in aquaculture. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?1 undergrad was trained during this period. How have the results been disseminated to communities of interest?We keep continuous communication with the TPWD catfish hatchery in San Marcos. We also contacted other catfish researchers and we are starting to write a new proposal to continue this research. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Objectives accomplished in year 3 Aim 1. Identify changes in catfish nasal and gut microbiomes exposed to several sub-acute concentrations of nitrite. We prepared two manuscripts that are close to submission. 1. Nicole Restrepo Caicedo*, Ashley Franklin**, Whitney Ortiz**, Mar Huertas and Camila Carlos-Shanley. Characterization of Genome Profiles found in Microbiome-derived Bacterial Isolates. To be submitted in Microbial Genomics. 2. Franklin, A**., Restrepo, N.*, Ortiz, W.**, Matthews, M., Prangnell, D., Carlos-Shanley, C. and Huertas, M. Development of nasal and gut probiotics to protect intensive catfish culture against Edwardsiella ictaluri infections. To be submitted in Journal of Experimental Biology. * Undergrad student,** Graduate students Aim 2. Develop a screening method to evaluate olfactory infection of E. ictaluri or ESC vaccine in catfish exposed to nitrites. We completed characterizing the main odorants in catfish and tested olfactory sensitivity of fish exposed to 0.2 mM nitrite and/or probiotic for two months. We analyzed all data and found that probiotics have a protective effect in nitrite exposure, and sensitivity to odorants remains similar to those control. Interestingly, fish exposed to nitrite can detect odorants at lower concentrations, but the olfactory signals in general were smaller than control and probiotic treatments. Thus, changes in the nose microbiome seems to be related with olfactory function and probiotic treatments can help to enhance the sense of smell. Aim 3. Identify and test potential nasal and gut probiotic strains to protect catfish against E. ictaluri in catfish reared with nitrites. We analyzed fish tissues biochemistry and histology from experiments designed in aim 3. Exposure to nitrite and probiotics treatments changed the physiology and anatomy of the nose and gut of catfish. Additionally, these treatments modulated the degree of infection of E. ictaluri and routes of infection. Nitrite accumulated during treatments, but after challenge with the pathogen, nose, gut and kidney tissues return to control values. Interestingly, brain nitrite values remained depleted after challenge in nitrite and/or probiotic treatments. Histological analysis we showed that even low concentrations of nitrite (0.2 mM) can damage fish nose and gut, but fish under probiotic treatment have less morphological alterations after exposure to nitrite. We analyzed pathological changesin nose and gut of fish exposed to E. ictaluri after nitrite and or probiotic treatment. We found that fish feed with probiotic have nos inflamatory procces due to the probiotic treatment and nitrite. However tissues were not altered by exposure to pathogen. Fish exposed to the pathogen in absence of probiotic had sign of bacterial infection and tissue damage in nose and gut. This damage was characterized by hyperthropia of the epithelia, hemorrage, white blood cell infiltration and cellular death. Changes in the microbiome due to nitrite treatments correlated with an increased susceptibility to E. ictaluri infections in intensive farming. In the long term, this research aims to provide guidelines for probiotic treatments in order to decrease the rate of bacterial infection and transmission in aquaculture system Additionally. We attended one conferences and communicated with interested parts in catfish culture. We continuously communicate with the Texas Park and Wildlife catfish hatcheries. We are writing a manuscript that includes results from aims 2 and 3

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Huertas, M. Franklin, A., Restrepo, N., Ortiz, W., Matthews, M., Prangnell, D., Carlos-Shanley, C. . 2024. Development of nasal and gut probiotics to protect intensive catfish culture against Edwardsiella ictaluri infections. Aquaculture America 2023
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Mar Huertas. 2024. The double-edged sword effects of nitrite, nitrate, and agricultural pollution in fish. Seminars at the Department of Biology. Texas State University.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Mar Huertas. 2024. A new tale about environmental pollution: Water-borne nitrite effects in the chemical senses of aquatic vertebrates. Kavli Institute Seminar. Norway. April 2024.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Mar Huertas. 2024. DO FISH HAVE NOSE? COVID, infections, pollutants, and other horror stories February. University of Insubria Seminar. Italy. February 2024


Progress 09/01/22 to 08/31/23

Outputs
Target Audience:We reached out to the catfish community during Aquaculture America 2023 (New Orleansmeeting) and The Society for Integrative and Comparative Biology (SICB) 2023meeting. In the Aquaculture America meeting, we interacted with both scientists and catfish farmers and shared information about our research. We further developed several collaborations in the identification of fish microbiomes with application to aquaculture practices in Texas and out of state. In the SICB meeting, we shared with the research community our advances in morphological changes in chemical senses due to nitrites, a common water pollutant, and the effects of probiotics in E. ictalluri infection. Nasal probiotics are a novel idea in aquacultureand the interactions with other vertebrate physiologists gave insights into the design of new treatments. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?1 Master students and 1 undergrad were trained during this period. The master student completed her thesis in November 2023. The master students attended two National conferences and both students attended one State conference. How have the results been disseminated to communities of interest?We keep continuous communication with the TPWD catfish hatchery in San Marcos. We also contacted other catfish researchers and shared our results in professional meetings. What do you plan to do during the next reporting period to accomplish the goals?We are finishing some histological and biochemical analysis and we are mostly working in the writing of two manuscripts. We also intend to use our data to apply to the next NIFA call including new co-Pi's from the States of Texas and Mississipi.

Impacts
What was accomplished under these goals? Nitrite is a nitrogenous waste derived from fish excretion that can accumulate in catfish intensive farms and increase the occurrence of enteric septicemia of catfish (ESC) caused by the bacteria Edwardsiella ictaluri. This pathogen can infect the gut and the nose of fish leading to disease and death, causing millions of dollars of loses in animal production. To date, prophylactic treatments to prevent E. ictaluri infections are nonexistent. However, prophylactic treatments with nasal and gut probiotics can give an additional protection against pathogens and nitrite damage. In the second year of this project, we sequenced the different probiotic strains we used in our probiotic treatments before pathogen challenge experiments. We performed the histological, biochemical and electrophysiological analysis after the pathogen challenge experiments in individuals formerly treated with probiotics and/or nitrite. Our long-term goal is to develop nasal and gut probiotics that can protect catfish against ESC in intensive culture systems with poor water quality. Objectives accomplished in year 2 Aim 1. Identify changes in catfish nasal and gut microbiomes exposed to several sub-acute concentrations of nitrite. We isolated the microbiome of catfish exposed to sub-acute concentrations of nitrite in the first year and sent it for sequencing. Twelve bacterial isolates used for disease mitigation in Channel catfish (Ictalurus punctatus) were classified and analyzed via DNA extraction, Genome sequencing and genome-web identifiers to gather the genomic profiles of each isolate including nomenclature name, genome total length (bp), contigs, gene cluster content%, number of biosynthetic gene clusters and type of biosynthetic gene clusters. Bacterial Isolates: 12 bacterial isolates were classified before DNA extraction. Each isolate was given a strain ID that was used for research purposes. In the Probiotic cocktail were present five different isolate classifications, Aeromonas Dhakensis, Aeromonas veronii, and Plesiomonas shigelloides are gram-negative bacteria derived from the fish's gut. Lactococcus pentauri, is a gram-positive bacteria derived from the fish's gut. During classification, a potential new species of Acinetobacter was described as a gram-negative bacillus found in the fish's nose. DNA Analysis: The bacterial isolate's DNA was extracted using plated aliquots of each isolate. Then the bacterial colonies found on the plates were analyzed using Qiagen Bacteremia DNA kit (following manufacturing protocol for extractions on bacteria cultures), and Wizard Genomic DNA purification kit (following manufacturing protocol for extractions on bacteria cultures). After DNA extraction was complete, the DNA concentration was measured using a Qubit4 Fluorometer, and lastly, all the isolate that measured a concentration of 2 ng/µL or more were sent to a commercial laboratory for 16s rRNA genomic sequencing. Online-Genmic Analysis: After the genomic sequencing was obtained from the commercial laboratory, two web servers, antiSMASH bacterial version and EzBioCloud ANI Calculator, were used to analyze the genomes information and compare them to existing database.The AntiSMASH server was used to gather information about each of the isolates Biosynthetic Gene Cluster (BGC), and total length (bp). EzBioCloud ANI Calculator was used to gather data on Contigs number, Genetic Custer content % (GC%), total length (bp) and OrthoANIu values of similarity between all of the isolates classified as Plesiomonas shigelloides. Summarizing, we described genomic data of bacterial isolates that were used for probiotic treatment for Channel catfish (Ictalurus punctatus) in aim 3. Using orthoANI calculator, the similarity value of isolates of classification Plesiomonas shigelloides was analyzed and appeared in most of the isolates with ninety six percent similarity. Characterization of this isolates can be used in aquaculture for probiotic-creation purposes and decrease diseases induced by pathogenic organisms. The results of sequencing have been collected in a publication that will be submitted shortly. Aim 2. Develop a screening method to evaluate olfactory infection of E. ictaluri or ESC vaccine in catfish exposed to nitrites. We completed characterizing the main odorants in catfish and tested olfactory sensitivity of fish exposed to 0.2 mM nitrite and/or probiotic for two months. We analyzed all data and found that probiotics have a protective effect in nitrite exposure, and sensitivity to odorants remains similar to those control. Interestingly, fish exposed to nitrite can detect odorants at lower concentrations, but the olfactory signals in general were smaller than control and probiotic treatments. Thus, changes in the nose microbiome seems to be related with olfactory function and probiotic treatments can help to enhance the sense of smell. Aim 3. Identify and test potential nasal and gut probiotic strains to protect catfish against E. ictaluri in catfish reared with nitrites. We analyzed fish tissues biochemistry and histology from experiments designed in aim 3. Exposure to nitrite and probiotics treatments changed the physiology and anatomy of the nose and gut of catfish. Additionally, these treatments modulated the degree of infection of E. ictaluri and routes of infection. Nitrite accumulated during treatments, but after challenge with the pathogen, nose, gut and kidney tissues return to control values. Interestingly, brain nitrite values remained depleted after challenge in nitrite and/or probiotic treatments. Histological analysis we showed that even low concentrations of nitrite (0.2 mM) can damage fish nose and gut, but fish under probiotic treatment have less morphological alterations after exposure to nitrite. We are currently analyzing pathological changes of fish exposed to E. ictaluri after nitrite and or probiotic treatment. Preliminary data indicates that probiotic fish had less morphological alterations than those exposed to nitrite.Alteration of the natural nasal and gut morphology by nitrite may increase infection rate E. ictaluri and probiotic treatments can reduce infection even if water quality is low (high nitrite content). Additionaly, we found that fish exposed to nitrite loose bacterial load in their nose and gut microbiomes. Whereas fish treated with probiotics increased bacterial load. Itis uncertain if the increase in bacterial load was also due to E. ictaluri presence. Changes in the microbiome could be the missing link between nitrite and increased susceptibility to E. ictaluri infections in intensive farming. In the long term, this research aims to provide guidelines for water quality management in order to decrease the rate of bacterial infection and transmission in aquaculture systems. Additionally. We attended three conferences and communicated with interested parts in catfish culture. We continuously communicate with the Texas Park and Wildlife catfish hatcheries. We are writing a manuscript that includes results from aims 2 and 3.

Publications

  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Ashley Franklin, Nicole Restrepo, Camila Carlos-Shanley, Mar Huertas (2023). Bacteria and nitrite change smelling ability of catfish. 2023 TXST Stem Conference. March 24th. San Marcos, Texas.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Characterization of Microbiome-derived Probiotics Used for Disease Mitigation in Channel Catfish. Nicole Aileen Restrepo Caicedo, Ashley Franklin, Mar Huertas Pau, Camila Carlos-Shanley. 2023 TXST Stem Conference. March 24th. San Marcos, Texas.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Ashley Franklin, Whitney Ortiz, Michael Matthews, David Prangnell, Camila Carlos-Shanley and Mar Huertas. The Impact of Nitrite and Edwardsiella ictaluri on Channel Catfish (Ictalurus punctatus) Nose Morphology. SICB 2023
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Ashley Franklin, Whitney Ortiz, Michael Matthews, David Prangnell, Mar Huertas and Camila Carlos-Shanley. Development of nasal probiotics for pathogen, edwardsiella ictaluri. Aquaculture America 2023
  • Type: Theses/Dissertations Status: Accepted Year Published: 2023 Citation: Ashley Franklin. (2023). Effects of nitrite and probiotics on physiology, olfaction, and disease susceptibility in channel catfish. Master of Science with a Major in Aquatic Resources. Texas State University.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Mar Huertas (2023). When the nose fails. Invited seminar speaker at Texas A&M San Antonio. Texas


Progress 09/01/21 to 08/31/22

Outputs
Target Audience:We reach out to the catfish community during Aquaculture America 2022 meeting and the comparative physiology research community during Experimental Biology 2022 meeting. In the Aquaculture America meeting, we interacted with both scientist and catfish farmers and share information about our research. We started several collaborations in the identification of fish microbiomes with application to aquaculture practices. In the Experimental Biology meeting, we shared with the research community our advances in morphological changes in chemical senses due to nitrites, a common water pollutant. The idea of physiological changes due to chronic exposure to non-lethal concentration of a pollutant was greatly apreciated. As a result we have been invited to write a review about this subject in the Aquaculture and Fisheries Journal. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?2 Master students and 1 undergrad were trained during this period. Both master students attended two National and International conferences. How have the results been disseminated to communities of interest?We keep continuous communication with the TPWD catfish hatchery in San Marcos. We also contacted other catfish researchers and shared our results in professional meetings. What do you plan to do during the next reporting period to accomplish the goals?We aim to send our samples for shotgun sequencing and finish the analysis of the experiments perfomed in each of the objectives and stablish new plans of action to design new probiotics. We also aim to reach further to the catfish farming community for future collaborations in upscaling probiotic usage.

Impacts
What was accomplished under these goals? Nitrite is a nitrogenous waste derived from fish excretion that can accumulate in catfish intensive farms and increase the occurrence of enteric septicemia of catfish (ESC) caused by the bacteria Edwardsiella ictaluri. This pathogen can infect the gut and the nose of fish leading to disease and death, causing millions of dollars of loses in animal production. To date, prophylactic treatments to prevent E. ictaluri infections are nonexistent. However, prophylactic treatments with nasal and gut probiotics can give an additional protection against pathogens and nitrite damage. In the first year of this project, we characterized the response of the catfish nasal and gut microbiomes to different concentrations of nitrite. Then, we identified nose and gut bacteria resilient to nitrite and pathogens (more than 95% of pathogen inhibition) and used to formulate a new probiotic mix. Finally, we fed fish with the newly formulated probiotic for two months and, after that, we exposed fish to the pathogen. The results of the pathogen challenge are under analysis. Our long-term goal is to develop nasal and gut probiotics that can protect catfish against ESC in intensive culture systems with poor water quality. Objectives accomplished in year 1 Aim 1. Identify changes in catfish nasal and gut microbiomes exposed to several sub-acute concentrations of nitrite. We performed all fish experiments and are currently working in the analysis of bacterial sequencing. We exposed catfish to acute (lethal) and sub-acute (sublethal) nitrite and collected samples for microbiome sequencing, biochemical and histological analysis. We isolated the microbiome and is sent for sequencing. Channel catfish were exposed to 0 mM (control), 0.2 mM, and 2 mM concentrations of nitrite. Atotal of six 500-liter tanks were used in a two-phase experiment. For phase one, 16 fish per tank were placed into three 0 mM nitrite tanks and three 2 mM nitrite tanks. The 2 mM fish were kept for 12 hours before sampling. For phase two, 24 of the phase one control fish were placed into three 0.2 mM tanks and the remaining 24 control fish were left in the three control tanks for a total of 8 fish per tank. Phase two lasted 30 days with sub-samplings at day five and 30. The mortality rates were 0.04%, 0.08%, and 92% for the control, 0.2 mM, and 2 mM treatments respectively. All fish were sampled for nose, gut, muscle, gill, brain, blood, liver, and kidney tissues for biochemical, microbial, or histological analysis. Upon dissection, fish from the 2 mM tanks exhibited brown blood disease, which is due to the nitrite-induced oxidation of hemoglobin. In phase two experiments (long term exposure to nitrite), fish did not show any visible signs of distress during culture and there were not symptoms of brown blood disease. However, tissue analysis showed oxidative damage in gut, nose and gill in individuals exposed to 0.2 mM treatment. Thus, long-term exposure to non-lethal nitrite can induce subtle negative effects in the catfish health. The nose and gut microbiome were extracted and used to culture potential probiotics or for RNA extraction. We found different bacterial grow in individuals exposed to nitrite compared to those control. RNA will be sequenced using whole genome shotgun sequencing to determine the microbial composition and measure changes in relative abundance. Aim 2. Develop a screening method to evaluate olfactory infection of E. ictaluri or ESC vaccine in catfish exposed to nitrites. We developed the electro-olfactogram recording (EOG) technique to measure olfactory response of catfish to food odorants and vaccine, prior and posterior to ESC infection. EOG experiments are currently ongoing. So far, we identified the amino acids and bile acids that elicit the most olfactory sensitive responses. The amino acids with strongest olfactory responses were serine and alanine, whereas the bile acids with the strongest responses were taurochenodeoxycolic acid and chenodeoxycholic acid. After characterizing the main odorants in catfish, we tested olfactory sensitivity of fish exposed to 0.2 mM nitrite and/or probiotic for two months. Preliminary experiments found that nitrite and probiotic alter olfactory function, with loss of sensitivity for some odorants in both groups. Thus, changes in the nose microbiome seems to be related with olfactory function Aim 3. Identify and test potential nasal and gut probiotic strains to protect catfish against E. ictaluri in catfish reared with nitrites. We identified 10 bacterial strains isolated in aim 1 with more than 95% in vitro inhibitory activity to the pathogen E. ictaluri. We produced a probiotic diet that we used to perform clinical experiments to pinpoint potential live probiotic that will protect catfish from E. ictaluri. We also run several lethal dose experiments to identify a strain of E. ictaluri that produce hemorrhagic symptoms in three days but doesn't kill all the fish, so we can take samples of tissues and microbiomes. We found that the best concentration of E. ictaluri in our laboratory was 1x 107 CFU/ml. For the clinical treatment, we exposed catfish for 2 months to either control water, nitrite (0.2 mM), probiotic or nitrite with probiotic. Fish didn't show sign of distress or mortality during that period. After that, fish were challenged for 3 days with E. ictaluri. Fish exposed to only nitrite showed more symptoms of E. ictaluri infection (ulcers and hemorrhages). Fish treated with probiotic didn't have nose infection by E. ictaluri. At the end of the challenge, we collected samples for microbiome sequencing, biochemical and histological analysis. We performed all the fish experiments in this objective, and we are currently analyzing catfish tissues and the microbiome. In conclusion, we performed all the fish experiments in the three objectives, and we are in our way to analyze fish tissues and microbiome sequencing data. We prioritized fish experiments because catfish juveniles are only available in late spring during catfish season. So far, our experiments showed that even low concentrations of nitrite (0.2 mM) can damage fish tissues and microbiomes after long exposure. These effects can happen without visual sign of fish distress. Because the microbiome is a powerful barrier against pathogens, disruption of the microbiome could lead to higher rates of disease infection and transmission in fish. Alteration of the natural nasal microbiome by nitrite may increase mortality from the bacteria that cause septicemia in catfish, Edwardsiella ictaluri, whose common routes of infection are the nose or gut. Changes to the microbiome could be the missing link between nitrite and increased susceptibility to E. ictaluri infections in intensive farming. In the long term, this research aims to provide guidelines for water quality management in order to decrease the rate of bacterial infection and transmission in aquaculture systems. Additionally. We attended to two aquaculture conferences and communicate with interested parts in catfish culture. We continuously communicate with the Texas Park and Wildlife catfish hatcheries. We published a paper about microbiomes affected by nitrites in goldfish. Although the experiments and funding are prior to this project the microbiomes associated with goldfish were used to design catfish probiotics.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Franklin, A., Ortiz, W., Matthews, M., Prangnell, D., Carlos-Shanley, C. and Huertas, M. (2022), Chronic Exposure to Sublethal Concentrations of Aquatic Nitrite Change Nose and Gut Morphology and Associated Microbiomes in Channel Catfish. The FASEB Journal, 36:. https://doi.org/10.1096/fasebj.2022.36.S1.R4523
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Whitney E. Ortiz, Mar Huertas, Camila Carlos-Shanley.(2022). Impact of sublethal concentrations of nitrite on goldfish (Carassius auratus) microbiomes. Microb Ecol. 2022 Oct 25. doi: 10.1007/s00248-022-02123-4. Epub ahead of print. PMID: 36282286.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Ashley Franklin*, Whitney Ortiz, Michael Matthews, David Prangnell, Camila Carlos-Shanley and Mar Huertas.(2022). Nitrite induces changes in the channel catfish nose and gut microbiome. WAS (World Aquaculture Symposium) 2022 San Diego. Oral Presentation.