Progress 09/01/20 to 08/31/23

Outputs
Target Audience:Our target audiences over the course of this USDA-NIFA funded project included: A) personnel that received scholarly scientific training and mentorship, including: i) three graduate students (Sean Lennox, Nisha Shrestha, and Sam Darling, Michigan State University) in PD-Loch's laboratory that received scholarly mentorship and training (and also mentored and trained others) in flavobacteriological, microbiological, and molecular techniques, data analyses, and effective scientific oral and written communication. Of particular note, PhD student Sean Lennox was recognized for substantial scientific achievements and highly effective oral communication when he received three "Best Student Presentation" awards for the presentations he gave on findings from this grant at the 17th Annual Department of Fisheries and Wildlife Graduate Student Organization Research Symposium (2022) and in both the 2022 and 2023 American Fisheries Society - Fish Health Section Virtual Seminar Series. ii) three undergraduate students (e.g., Brady Yokom, Giuseppe Cavaliere, and Christine Smeltzer) that received training in a range of microbiological laboratory techniques, diagnostic testing, aquatic animal husbandry, and experimental design. Notably, all three students have now either been accepted or are applying to veterinary or graduate school. iii) two postdoctoral researchers (Dr. Megan Shavalier, Dr. Lori Ivan) that played integral roles in this study; B) undergraduate, pre-veterinary, and graduate students enrolled in PD Loch's Principles of Fish and Wildlife Disease lecture and laboratory courses; C) attendees of professional scientific meetings (e.g., the 2022 and 2023 national American Fisheries Society - Fish Health Section Summer Seminar Series, the 2022 International Symposium of Aquatic Animal Health, the Annual MSU Department of Fisheries and Wildlife Graduate Student Organization Research Symposia, the national 46th Eastern Fish Health Workshop; D) members of the Great Lakes Fishery Commission - Great Lakes Fish Health Committee (with members from eight Great Lakes states and two Canadian provinces); E) members and stakeholders of USDA's North Central Regional Aquaculture Center (NCRAC); F) fishery biologists and technicians employed at multiple aquaculture facilities and state fish hatcheries in the USA; G) fish health professionals and aquatic animal veterinarians); k) members of PD-Loch's laboratory during regularly scheduled laboratory meetings; H) students enrolled in Conservation Medicine (FW 821, MSU); I) members of the Northeast Fish Health Committee representing 13 US states plus the District of Columbia; J) international veterinary students and other attendees (n>800) of the "Three in One" Visiting Scholar Program (organized by Brawijaya University, East Java, Indonesia) hailing from multiple countries in Southeast Asia and Europe; K) international attendees of the 4th International Conference On One Health (ICOH). Our efforts to deliver science-based knowledge that resulted from this USDA-NIFA funded project include: a) multiple oral presentations (both virtual, recorded, and in-person) that were given by PD Loch and Mr. Sean Lennox (i.e., the graduate student whose research was funded, in part, by this USDA-NIFA project) at regional, national, and international professional conferences and meetings; b) outreach via discussions and meetings with and presentations to fishery managers, fishery biologists, and aquaculture facility personnel (e.g., through group meetings at hatcheries, through presentations to Michigan Department of Natural Resources Fisheries Division, through discussions with members of the Great Lakes Fishery Commission - Great Lakes Fish Health Committee; through discussions with producers and industry, including at the 2020 NCRAC meeting); c) laboratory instruction, training, mentorship, discussions, and experiential learning for multiple students (graduate and undergraduate); d) formal classroom instruction (in person) for Michigan State University graduate and undergraduate students enrolled in PD-Loch's Principles of Fish and Wildlife Disease lecture and laboratory courses; e) lectures, case studies, and discussions in AQUAVET® II and FW 894 (Principles and Perspectives in Fisheries and Wildlife, MSU); f) in dissertation chapters (n=4) and manuscripts (n=4) that are currently in preparation; g) published conference proceedings; and h) sharing of our LAMP assay and protocols with an industry partner towards eventual commercialization of this "pondside" test for use by industry and natural resource agencies. Following completion of this USDA-NIFA funded study, nine people (eleven when counting the PD/Co-PDs) received experiential laboratory learning, training, and mentor/menteeship over the course of this study (e.g., three undergraduate students, three graduate students, two post-doctoral researcher, and one international scholar). In total, 19 presentations at professional conferences and meetings were given, with several more to be given in 2024. Similarly, >25 meetings and discussions took place between the PD/project personnel and fish hatchery staff to discuss the findings from this USDA-NIFA funded research. It is also noteworthy that this USDA-funded research supported the majority (i.e., four) chapters comprising the in-preparation dissertation of PhD student Sean Lennox, along with at least four in preparation manuscripts. Changes/Problems:Initial pandemic related challenges led to removal of originally proposed subtasks 3a and 3b and subtask 8b, which was approved by USDA-NIFA. What opportunities for training and professional development has the project provided?Stemming directly from this USDA-NIFA funded study, numerous training and professional development opportunities were afforded to a multitude of students and scientists. Nine people hailing from a diversity of backgrounds and a range of life experiences received mentorship, training, and opportunities for scientific scholarly growth over the course of this study, including three undergraduate students at Michigan State University (MSU), three graduate students (MSU), two postdoctoral researchers (MSU), and a visiting international adjunct scholar from the University of Sao Paulo State (Sao Paulo, Brazil). Training opportunities included gaining substantial experience in technical laboratory skills (e.g., in vitro and in vivo bacteriological techniques, molecular assay design and optimization, bioinformatics,) to immersive experiences at fish (i.e., field) facilities to experiences in effective written and oral scientific communication and scholarly development. Further developing and skills and expertise in the design of controlled laboratory experiments, molecular assay development and testing, data collection, and analysis were focused upon heavily. Trainees were also integral to experimental design, experiment completion, data collection and interpretation, and were engaged in communication of findings to hatchery and aquaculture facility personnel, as well as a range of other stakeholders (researchers, clinicians/aquatic veterinarians and fish health professionals). Notably, a research assistantship from this USDA-NIFA grant provided partial support for PhD student Sean Lennox (MSU), who is on track to defend his dissertation by at least the Summer of 2025. The investment by USDA-NIFA in this project provided numerous opportunities for Sean's scholarly and scientific growth, and a sizeable portion of the resultant experiments and findings are included in his in-progress dissertation. Also of importance, Mr. Lennox embraced an impressive mentorship role for other students (both graduate and undergraduate alike), thereby fostering his own scholarly mentorship skills. Our team would also like to highlight that one of the undergraduate students that received training as a part of this grant applied to and has been accepted into the graduate program at MSU and will be pursuing a Master's degree focused on aquatic animal health in PD Loch's lab beginning in January 2024. Likewise, another undergraduate student receiving mentorship and training during this USDA-NIFA funded study has applied to veterinary school (with interests in aquatic animal health) and the third is also applying to graduate schools for 2024 (again with interest in aquatic animal health). Clearly, the initial investment made here by USDA-NIFA is fostering the development of the next generation of aquatic animal health professionals. In addition to the nine mentees/trainees noted above, further learning and training opportunities were made possible, in part, by this study for undergraduate and graduate students enrolled in PD Loch's Principles of Fish and Wildlife Disease lecture and laboratory courses (Fall semester 2021, n=21 students; Fall semester 2023, 15 students), and for other members of PD Loch's lab as this project and the associated experiments were discussed, planned, and completed. Although COVID-19 initially hampered attendance of in-person conferences and workshops, Professional Development opportunities that resulted from this USDA-NIFA funded project included substantial development of effective written and oral scientific communication skills for PhD student Lennox, who gained invaluable experience while authoring professional scientific abstracts, writing multiple chapters for his dissertation (also in preparation for submission to peer-reviewed journals), and presenting his research findings in multiple professional meetings and conferences. In this vein and as testament to his continued success in scientific communication, Sean was awarded three "Best Student Presentation" awards for his oral presentations of findings from this USDA-NIFA funded study that were given at professional regional (e.g., 17th Annual Department of Fisheries and Wildlife Graduate Student Organization Research Symposium, 2022) and national (e.g., the 2022 and 2023 American Fisheries Society - Fish Health Section Virtual Seminar Series) scientific venues. Additional professional development opportunities included attendance and presentation of findings by our study team at eighteen local, regional, national, and international scientific meetings, workshops, and professional conferences, including the 2021, 2022, and 2023 American Fisheries Society - Fish Health Section Virtual Seminar Series; the 16th, 17th, and 18th Annual Department of Fisheries and Wildlife Graduate Student Organization Research Symposium, the 46th Annual Eastern Fish Health Workshop; the 9th International Symposium of Aquatic Animal Health, Santiago, Chile; please see "Products" section for additional meetings and presentations given). Likewise, the substantial laboratory and fish health/husbandry/diagnostic/bioinformatic expertise that have been gained by students involved in this study, as well as the growth of involved graduate students as scholarly mentors of the next generation of scientists resulting from the opportunities that were provided by this USDA-NIFA funded project cannot be understated. How have the results been disseminated to communities of interest?New knowledge and results generated during this USDA-NIFA funded project were disseminated extensively to a range of stakeholders and communities of interest through multiple avenues, including via: a) nineteen professional oral presentations at regional (n=10), national (n=5), and international (n=4) scientific conferences and meetings - notably, three presentations by Mr. Lennox that conveyed findings from this USDA-NIFA funded study were recognized with Best Student Presentations Awards; b) four didactic university courses (e.g., two lecture-based, two laboratory-based; Principles of Fish and Wildlife Disease lecture and laboratory) that are taught by PD-Loch and attended by undergraduate, pre-veterinary, and graduate student enrollees; c) discussions and lectures on fish-pathogenic flavobacteria given by the PD in AQUAVET® II (June 2021, virtual, June 2022; June 2023), a summer course for veterinary students interested in aquatic animal medicine from all over the USA; d) formal and informal discussions with fish farmers, agriculture and natural resources administrators, fish health professionals, aquatic veterinarians, academicians, regulators, students, and fish health committee members; e) formal and informal discussions with a range of aquaculture and natural resource manager personnel and fish farmers, including as our newly developed LAMP assay was being deployed into the field for pilot testing; f) discussions and presentations in regular lab meetings amongst the PDs research group; g) personal communications (via email, Zoom/Teams, phone calls, in-person) with producers, natural resource managers, fish health professionals, aquatic veterinarians, researchers, and students inquiring about flavobacterial fish diseases, associated research, and advice on the prevention and control of flavobacterial infections in fish; h) in-class (e.g., FW 821, MSU) discussions for effectively communicating science towards real-world benefits by Mr. Lennox; i) research updates to MSU faculty members serving on Mr. Lennox's Graduate Committee; j) f) discussions with USDA-NIFA's North Central Regional Aquaculture Center (NCRAC) members and additional fish producers who attended the 2020 North Central Region Aquaculture Roundtable Listening Session (Columbus, OH) - notably, findings from this study were instrumental in informing a successfully funded grant proposal from NCRAC, focused on developing vaccine preparations again Fp in the North Central Region of the USA (project ongoing); k) sharing of our new LAMP assay and associated protocols with an Industry Partner, which resulted in a collaborative USDA-SBIR grant towards potentially commercializing our new Fp LAMP assay (project ongoing); l) in PD Loch's Promotion and Tenure Seminar in Fall 2023. Findings are also in the process of being further disseminated via scientific manuscripts that are presently in preparation (n=4), which will be submitted to peer-reviewed journals for publication (and ideally published as Open-Access and via additional support from MSU). Moreover, we anticipate additional presentations resulting from this USDA-NIFA funded project at upcoming fish health meetings in 2024 (e.g., Eastern Fish Health Workshop, MS; Great Lakes Fish Health Committee Summer Meeting, location TBD). What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Impact statement: Flavobacterium psychrophilum (Fp), causative agent of bacterial coldwater disease (BCWD), is a top disease impediment to trout and salmon production in the USA. Fp is readily transmitted from infected fish to fish, and from infected parents to offspring (i.e., vertical transmission) via reproductive fluids and eggs, making BCWD prevention and control difficult. Our other recent USDA-NIFA funded research uncovered substantial US Fp genetic diversity while revealing certain Fp strains are behind most BCWD outbreaks in the USA. Moreover, we showed Fp transmission from parents to offspring is a major source of such outbreaks, a concern given that BCWD vaccines are currently unavailable and current fish egg disinfection protocols are inconsistent at best. Towards a lack of vaccines in the USA, we analyzed the serotypic (i.e. how fish immune systems "see" Fp during infections) diversity of >320 US Fp isolates recovered over several decades. Among others, interesting trends by fish host species were revealed (i.e. nearly 2/3 of rainbow trout isolates were molecular types (mtypes) 2 (35%) & 1 (31%), ~90% of Fp isolates from coho salmon were mtype 0), which not only will inform future vaccine development, but also guided development of a new loop-mediated isothermal amplification (LAMP) assay that is capable of detecting all currently recognized Fp molecular sero-variants and all tested US genovariants (n=116). This new Fp LAMP assay was optimized for eventual "pondside" use, requiring only simple equipment, and currently can rapidly and accurately detect Fp in "spiked" reproductive fluids <1 hour of sample collection - an invaluable tool for guiding the future use of "Fp-free" eggs for rearing. Experiments examining the efficacy of pre-fertilization egg disinfection revealed no Fp in eggs or hatchlings receiving our new disinfection method despite being exposed to Fp. A newly developed individual-based model showed increased fish survival across simulated rearing cycles following use of the new egg disinfection method and now serves as a means to forecast how changes in management practices will affect fish health and disease-associated losses. Strong positive impacts on the scholarly scientific development of multiple students (graduate and undergraduate) also resulted from the training, mentorship, and professional development opportunities that directly resulted from this USDA-NIFA funded study. This new knowledge, tools, and expertise for breaking Fp vertical transmission resulting from USDA-NIFA funding will certainly improve US-farmed trout and salmon productivity while also enhancing food security and hatchery-reared fish health. Objective 1- To identify the predominating Fp serotypes in the USA. Task 1: Serotyping of US-Fp isolates. Analyses of generated multiplex PCR assay data for 324 previously multilocus sequence typing (MLST) typed US-Fp isolates (+1 Canadian isolate) revealed many interesting trends, including by host fish species [e.g. ~2/3 of rainbow trout isolates were molecular type (mtype) 2 (35.3%) and 1 (30.5%), whereas ~90% of coho salmon Fp isolates were mtype 0]. Other uncovered trends of interest include that isolates belonging to clonal complex (CC) sequence type (ST) 10 were mostly mtype 1 and mtype 2, and no CC-ST10 isolates belonged to mtype 4. Objective 2- To develop & test a rapid assay for screening trout/salmon gametes that can be used "pondside." Task 2: Development of Fp LAMP assay. After developing new LAMP primers, loop primers were designed to increase efficiency of the LAMP reaction. In silico analyses showed compatibility with all known US Fp geno/sero-variants. Multiple nucleic acid extraction methods (e.g., boiling, microwaving) were pursued, whereby boiling proved promising in Fp "spiked" reproductive fluid. Substantial efforts went into optimization of the new Fp LAMP assay, with eventual incorporation of reagents that not only enhanced visual interpretation of results, but also reduced false positive risk and assay time from 60 to ~25 min. The new LAMP assay successfully detected Fp in "spiked" ovarian fluid (OF) and milt samples following a dilution step. Task 3 & 4: Evaluation of Fp LAMP sensitivity & specificity under lab & field conditions. Specificity of the new LAMP assay proved high, with no reactivity to >20 non-target bacterial fish pathogens despite 116 US Fp genetic variants yielding positive results. When run on triplicate serial dilutions of Fp ranging from 100,000-1 copy per reaction in OF and 8000-0.08 copies per reaction in milt, the analytical limit of detection was estimated at ~10 and ~80 copies, respectively. The new LAMP was also deployed under field conditions on Atlantic salmon and rainbow trout reproductive fluids, with a subset showing reactivity. Objective 3- Test & optimize a novel iodophor-based approach for salmon/trout egg disinfection. Task 5: Evaluating efficacy of iodophor egg disinfection pre-fertilization. Pilot experiments were conducted to verify that transportation followed by fertilization at our lab would yield suitable hatch rates, which ranged from 42-76%. The same process was followed for full experiments. Both pilot and full-scale Fp egg challenge and disinfection experiments were undertaken; in the full (n=12 treatment groups, in duplicate), half were immersed in Fp (6x108 CFUs/ml) and half were unexposed. Treatment groups included pre-fertilization treatment with 50ppm iodophor in saline, sterile saline only, or no treatment and water hardening treatment of either 100ppm iodophor or water only. A subset of dead eggs, dead fry, and live individuals of multiple life stages were assayed for Fp via culture (using a highly sensitive new Fp culture medium developed in our lab in another USDA-NIFA funded study) and PCR analyses of recovered bacteria. Fp was not detected in the unexposed groups, nor in the 50ppm iodophor pre-fertilization treatment group (compared to Fp detections at a ~2% to ~47% prevalence in the other Fp-exposed treatments), yielding very promising results for the trout and salmon industry. Task 6: Assessing efficacy of the optimized iodophor egg disinfection for eliminating Fp in facilities. This promising pre-fertilization egg disinfection method is being adopted and evaluated in ongoing collaborations with industry/agency partners. Objective 4- Enhance best management practices for preventing BCWD via modeling. Task 7: Individual-based modelling & analyses. We developed an individual based model (IBM) that tracks eggs and fry survival assuming initial infection rates derived from objective 3 results. The model can examine various hatchery practices and disease assumptions to better understand and manage fish health in hatchery/farm settings, including disease progression in older fish, as well as changes in flow rates, removal of dead fish/eggs, systems, and treatment, as well as differences in disease dynamics (e.g., mortality, infection rates). Results from the model show that fish survivors across simulated rearing cycles increased substantially following use of the new egg disinfection method. This new IBM represents an innovative way to forecast how changes in management practices will affect fish health and disease-associated losses. Objective 5- To disseminate enhanced BMPs & training. Task 8: Provide engaging outreach and training for hatchery & aquaculture personnel. The findings and recommendations resulting from this USDA-NIFA funded project were presented by the PD and team during multiple webinars and in-person workshops and meetings (see training and dissemination sections below). In all cases, these learning opportunities were beneficial, based upon very positive feedback from stakeholders and via 3 "Best Student Presentation" awards by PhD student Sean Lennox as he presented his USDA-NIFA funded findings.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Lennox SMG, Shavalier MA, Brenden TO, Knupp CK, Call DR, Soto E, Zhang Q, Loch TP. Developing a Novel Field-Based Assay and Improved Egg Disinfection Methods to Prevent Bacterial Coldwater Disease in Trout and Salmon. Presented at the 18th Annual Michigan State University Department of Fisheries and Wildlife Graduate Student Research Symposium, East Lansing, MI, 2023.
• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Lennox SMG, Shavalier MA, Brenden TO, Knupp CK, Loch TP. Developing an Improved Egg Disinfection Method to Reduce Flavobacterium psychrophilum Transmission Risk in Rainbow Trout (Oncorhynchus mykiss). Presented virtually at the American Fisheries Society  Fish Health Section Summer Seminar Series, 2023. Best Student Presentation Award
• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Lennox SMG, Shavalier MA, Brenden TO, Knupp CK, Call DR, Soto E, Zhang Q, Loch TP. Preventing Bacterial Coldwater Disease by Breaking Vertical Transmission of Flavobacterium psychrophilum. Invited Presentation at the Michigan Dept. of Natural Resources Fish Production Section Meeting, East Lansing, MI, 2023.
• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Lennox SMG, Shavalier MA, Brenden TO, Knupp CK, Call DR, Soto E, Zhang Q, Loch TP. Characterizing the Serotypic Diversity of Flavobacterium psychrophilum in Michigan. Presented at the Michigan Dept. of Natural Resources Fish Production Section Meeting, East Lansing, MI, 2023.
• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Knupp CK, Lennox SMG, Call D, Soto E, Shavalier MA, Shrestha N, Faisal M, & Loch TP. Flavobacteria: An Emerging and Resurging Threat to Fish Health Worldwide. Invited Seminar for the Egyptian Aquatic Health Association Monthly Seminar series, Cairo, Egypt, 2023.
• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Lennox SMG, Shavalier MA, Brenden TO, Knupp CK, Call DR, Soto E, Zhang Q, Loch TP. Developing a Novel Field-Based Assay and Improved Egg Disinfection Methods to Prevent Bacterial Coldwater Disease in Trout and Salmon. Presented virtually at the 46th Annual Eastern Fish Health Workshop, Atlantic Beach, NC, 2023.
• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Knupp CK, Lennox SMG, Call D, Soto E, Ivan L, Brenden T, Shavalier M, Shrestha N, Loch TP. Prevention and Control of Bacterial Coldwater Disease: Perspectives from Michigan. Invited Virtual Presentation for USFWS National Hatchery System Guest Webinar, 2023.
• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Knupp CK, Lennox SMG, Call DG, Soto E, Brenden TO, Shavalier MA, Loch TP. Enhancing Bacterial Coldwater Disease Prevention and Control by Uncovering the Diversity and Ecology of Flavobacterium psychrophilum. Invited Presentation in the USDA-NIFA Session for Projects Supporting Aquaculture, Aquaculture America, New Orleans, LA, 2023.

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

Outputs
Target Audience:Our target audiences during the current reporting period included: a) one graduate student (Mr. Sean Lennox, Michigan State University) in PD-Loch's laboratory that received scholarly mentorship and training (and also mentored and trained others) in flavobacteriological, microbiological, and molecular techniques, data analyses, and effective scientific communication; b) three undergraduate students who received training in a multitude of microbiological laboratory techniques, aquatic animal husbandry, and experimental design; c) undergraduate, pre-veterinary, and graduate students enrolled in PD Loch's Principles of Fish and Wildlife Disease lecture and laboratory courses (Fall semester, 2021; 21 students); d) attendees (e.g., university graduate and undergraduate students, MSU faculty and staff, stakeholders from various regional fisheries management agencies) of the 17th Annual Department of Fisheries and Wildlife Graduate Student Organization Research Symposium; e) members of the Great Lakes Fishery Commission - Great Lakes Fish Health Committee (with members from eight Great Lakes states and two Canadian provinces); f) attendees of the national virtual American Fisheries Society - Fish Health Section Summer Seminar series; g) international attendees of the 9th International Symposium of Aquatic Animal Health (both virtual and in-person) in Santiago, Chile; h) upper management for the Michigan Department of Natural Resources Fisheries Division; i) fishery biologists and technicians employed at multiple aquaculture facilities and state fish hatcheries in the USA; j) fish health professionals and aquatic animal veterinarians); k) members of PD-Loch's laboratory during regularly scheduled laboratory meetings; l) students enrolled in Conservation Medicine (FW 821, MSU). Our efforts to deliver science-based knowledge that resulted from this ongoing USDA-NIFA funded project during the current reporting period include: a) multiple oral presentations (both in-person and virtual) at local, regional, national, and international professional conferences and meetings that were given by PD-Loch and Mr. Sean Lennox (i.e., the graduate student whose research is funded by this USDA-NIFA project); b) outreach and extension via discussions with and presentations to fishery managers, fishery biologists, and aquaculture facility personnel (e.g., through group meetings at hatcheries, through presentations to Michigan Department of Natural Resources Fisheries Division, through discussions with members of the Great Lakes Fishery Commission - Great Lakes Fish Health Committee); c) laboratory instruction, training, mentorship, discussions, and experiential learning for multiple students (graduate and undergraduate); d) formal classroom instruction (in person) for Michigan State University graduate and undergraduate students enrolled in PD-Loch's Principles of Fish and Wildlife Disease lecture and laboratory courses; e) lectures, case studies, and discussions in AQUAVET® II and FW 894 (Principles and Perspectives in Fisheries and Wildlife, MSU); f) in dissertation chapters and manuscripts that are currently in preparation. Changes/Problems:The continued challenges posed by the COVID-19 pandemic have resulted in some delays from the originally proposed project timeline. However and with the no-cost extension, we anticipate the objectives and tasks will be completed. What opportunities for training and professional development has the project provided?During the current reporting period of this ongoing USDA-NIFA funded study, numerous training and professional development opportunities have been (and continue to be) afforded to multiple individuals. Training opportunities have included: a) extensive training and mentorship for one graduate student (Mr. Sean Lennox) in PD-Loch's laboratory resulting from immersive, hands-on experience with flavobacteriological/microbiological/molecular laboratory experiments, data analyses, and activities for enhancing effective oral and written scientific communication skills. Importantly, Mr. Lennox has also taken on an impressive mentorship role for other students, graduate and undergraduate alike, thereby fostering his own scholarly mentorship skills. Likewise, Sean is currently on a research assistantship partially funded by this USDA-NIFA grant, and thus has learned and received training during several graduate level courses towards fulfillment of his graduate degree. Moreover, Sean's development as an effective communicator of science is garnishing prestigious recognition, as he received two "Best Student Presentation" awards at two professional meetings for his oral presentations on findings resulting from this USDA-NIFA funded study to date; b) training and mentorship for three undergraduate students (Brady Yokom, Christine Smeltzer, Giuseppe Cavaliere) in a range of microbiological laboratory techniques, aquatic animal husbandry, and experimental design; c) discussions and critical thinking related to pathogen diversity and the development of animal disease prevention and control strategies for undergraduate and graduate students enrolled in PD Loch's Principles of Fish and Wildlife Disease lecture and laboratory courses (Fall semester, 2021; 21 students); d) exercises in experimental design, data interpretation, and data analyses for members of PD-Loch's laboratory as experiments, results, and analyses to date were discussed and critical thinking sought; and d) additional opportunities for effectively communicating science towards real-world benefits by Mr. Lennox in courses (e.g., FW 821, MSU), communication with hatchery/aquaculture staff, and others. Although COVID-19 continues to hamper attendance of in-person conferences and workshops, Professional Development opportunities that have resulted from this USDA-NIFA funded project to date included: a) enhanced development of effective written and oral scientific communication skills for Mr. Lennox, who gained invaluable experience while authoring additional professional scientific abstracts, writing multiple chapters for his dissertation (which will also be submitted for peer-reviewed publications), and presenting his research findings in multiple professional meetings and conferences; b) participation of students involved in this study in multiple professional virtual seminars and conferences (e.g., the 2022 American Fisheries Society - Fish Health Section Virtual Seminar Series on fish health, the17th Annual Department of Fisheries and Wildlife Graduate Student Organization Research Symposium); c) the substantial laboratory and fish health/husbandry/diagnostic/bioinformatic expertise that have been gained by students involved in this study; and d) growth of involved graduate students as scholarly mentors of the next generation of scientists. How have the results been disseminated to communities of interest?New knowledge and results generated during this ongoing USDA-NIFA funded project have already been extensively disseminated to communities of interest through multiple avenues, including via: a) five professional oral presentations at local (n=2), regional (n=1), national (n=1) and international (n=1) scientific conferences and meetings (n=3 by the PD; n=2 by graduate student Sean Lennox). Of note, both presentations by Mr. Lennox that conveyed results to date from this USDA-NIFA funded study were recognized with Best Student Presentations Awards; b) two didactic university courses (e.g., one lecture-based, one laboratory-based) that are taught by PD-Loch and attended by undergraduate, pre-veterinary, and graduate student enrollees; c) discussions and lectures given by the PD in AQUAVET® II (June 2022), a summer course for veterinary students interested in aquatic animal medicine from all over the USA; d) formal and informal discussions with academicians, agriculture and natural resources administrators, fish health professionals, aquatic veterinarians, students, and fish health committee members; e) formal and informal discussions with a range of aquaculture and natural resource manager personnel, including as our newly developed LAMP assay was being deployed into the field for pilot testing; f) discussions and presentations in regular lab meetings amongst the PDs research group; g) personal communications (via email, Zoom/Teams, ResearchGate, phone calls, in-person) with producers, natural resource managers, fish health professionals, aquatic veterinarians, researchers, and students inquiring about flavobacterial fish diseases, associated research, and advice on the prevention and control of flavobacterial infections in fish; h) in-class (e.g., FW 821, MSU) discussions for effectively communicating science towards real-world benefits by Mr. Lennox; and i) research updates to MSU faculty members serving on Mr. Lennox's Graduate Committee. What do you plan to do during the next reporting period to accomplish the goals?Some delays continue to be experienced due to the ongoing pandemic, but the proposed objectives and tasks will be completed prior to the end of this project.

Impacts
What was accomplished under these goals? Impact statement: Flavobacterium psychrophilum (Fp), causative agent of bacterial coldwater disease (BCWD), is a top disease impediment faced by producers of trout and salmon across the USA. This microbe is readily transmitted from infected fish to fish, but also from infected parents to offspring (i.e., vertical transmission) via reproductive fluid and egg-associated Fp transmission, making BCWD prevention and control difficult. Our other recent USDA-NIFA funded research has uncovered substantial US Fp genetic diversity while revealing certain Fp strains are behind most BCWD outbreaks in the USA. Moreover, we have shown that Fp transmission from parents to offspring is a major source of such outbreaks, a concern given that BCWD vaccines are currently unavailable and that current fish egg disinfection protocols are inconsistent at best. In the vein of unavailable vaccines in the USA, our research team continued analyzing the serotypic (i.e. how fish immune systems "see" Fp during infections) diversity of 324 US Fp isolates (+1 isolate from Canada) recovered over several decades. Among others, interesting trends by fish species host were revealed (i.e. nearly 2/3 of rainbow trout isolates thus far are molecular types (mtypes) 2 (35%) & 1 (31%), ~90% of Fp isolates from coho salmon are mtype 0), which not only will inform future vaccine development, but also guided the development of a new loop-mediated isothermal amplification (LAMP) assay that is capable of detecting all currently recognized Fp molecular sero-variants. Of note, our new Fp LAMP assay is being further optimized for eventual "pondside" use, requiring only inexpensive/simple equipment (e.g. a heat block, pipette), and as of now, can rapidly and accurately detect Fp in "spiked" reproductive fluids <1 hour following reproductive fluid collection. This tool will be invaluable for guiding the use of "Fp-free" eggs for rearing. Complementarily, we completed pilot egg disinfection experiments and promisingly, less Fp infections were detected in eggs that were disinfected with the new protocol and no Fp was recovered from the resulting hatchlings ("fry"). Moreover, strong positive impacts on the scholarly scientific development of multiple students (graduate and undergraduate) has resulted from the training, mentorship, and professional development opportunities that directly resulted from this USDA-NIFA funded study. As this study finishes in the upcoming year, the newly generated knowledge, tools, and expertise for breaking Fp vertical transmission will certainly improve US-farmed trout and salmon productivity while simultaneously enhancing food security and hatchery-reared fish health. Objective 1- To identify the predominating Fp serotypes associated with economic losses in the USA. Task 1: Serotyping of US-Fp isolates. Further analyses of generated multiplex PCR assay data for 324 previously multilocus sequence typing (MLST) typed US-Fp isolates (+1 Canadian isolate) revealed interesting trends by host fish species, including ~2/3 of rainbow trout isolates thus far are molecular type (mtype) 2 (35.3%) and 1 (30.5%), whereas ~90% of coho salmon Fp isolates were mtype 0. Other uncovered trends of interest include that isolates belonging to clonal complex (CC) sequence type (ST) 10 have thus far primarily been mtype 1 and mtype 2, and no CC-ST10 isolates belonged to mtype 4. Additional trends are now being explored. Objective 2- To develop and test a rapid assay for screening trout/salmon gametes for all known US-Fp variants that hatchery biologists/farmers can use "pondside." Task 2a: Development of Fp qLAMP primers. After developing the required set of four LAMP primers, optional loop primers were designed to increase speed and specificity of the LAMP reaction. Sequence analysis revealed that a single base pair mutation occasionally occurred within the binding site of the LB primer; thus, a degenerate base was implemented and based on in silico analyses, ensured compatibility to all known US Fp geno/sero-variants. Task 2b: Nucleic acid (NA) extraction & optimization for field. Following previous progress, 2 simple NA extraction methods (boiling & microwaving) were pursued. Microwaving appeared to inhibit LAMP color change, but boiling (10 min) proved promising in Fp "spiked reproductive fluids (2d). Task 2c: Development and optimization of Fp LAMP & qLAMP assay. Implementation of WarmStart® Colorimetric LAMP 2X Master Mix with UDG from New England Biolabs not only further improved the ease of interpretation of our new LAMP assay while reducing the risk of false positives due to carryover contamination, but also reduced assay time from 60 to ~25 min. Specificity has proven to be high (i.e., 18 non-target bacterial fish pathogens did not result in positive results) and pilot testing in the field proved feasible and promising, with further analysis and optimization underway. Task 2d: Evaluation of Fp qLAMP on "spiked" gamete samples. The new LAMP assay successfully detected Fp in "spiked" ovarian and seminal (milt) samples following a dilution step. Task 3: Evaluation of qLAMP diagnostic sensitivity and specificity. LAMP was performed on serial dilutions of Fp ranging from 100,000 to 1 one copy per reaction in ovarian fluid (OF) and 8000 to 0.08 copies per reaction in milt (in triplicate) to assess the analytical limit of detection (LOD). Initial assessments suggest the LOD is ~10 copies per reaction in OF ~80 copies per reaction in milt, with further analyses underway. Task 4: Field evaluations of Fp qLAMP diagnostic sensitivity and specificity. The new LAMP has been deployed under field conditions on three occasions, (n=78 reproductive fluid samples from Atlantic salmon and rainbow trout), where 3 samples yielded weak positives. Further analysis and optimization is underway. Objective 3- To test and optimize a novel iodophor-based approach for salmon/trout egg disinfection that is effective against all US-predominating Fp sero/genotypes. Sub-task 5a: Origin of gametes for experiments. A pilot experiment of two groups was conducted to verify that transporting trout eggs followed by fertilizing them at our laboratory would yield suitable hatch rates, which ranged from 42-76%. Sub-task 5b: Fp challenge of eggs and iodophor disinfection experiments. A pilot Fp challenge and disinfection experiment was undertaken, consisting of four treatment groups that either were immersed in Fp at ~1 x 1010 CFUs/ml-1 or received no bacteria and received prefertilization disinfection or not. A subset of dead eggs was assayed for Fp via culture (using a highly sensitive new Fp culture medium developed in our lab in another USDA-NIFA funded study) and subsequent PCR analyses of recovered bacteria. No Fp was detected in the unchallenged groups; however, Fp was detected in ~20% of the pre-fertilization-disinfected + Fp challenged eggs compared to ~60% of the of the undisinfected + Fp challenged eggs. Promisingly and after hatching, 20 fry from each treatment were tested for Fp, revealing Fp infections only in the Fp challenged undisinfected eggs. The full-scale experiment is slated to begin early in the next reporting period. Task 6: Assessing efficacy of the optimized iodophor egg disinfection method for eliminating Fp in Atlantic salmon and rainbow trout facilities. To begin during next reporting period. Objective 4- To enhance best management practices (BMPs) for preventing/controlling BCWD via modeling of newly derived data. To begin during next reporting period. Objective 5- To disseminate enhanced BMPs and training to perform "pondside" testing to aquaculture/hatchery personnel via workshops and webinars. To occur following completion of Obj.1-4.

Publications

• Type: Conference Papers and Presentations Status: Other Year Published: 2022 Citation: Knupp CK, Lennox SMG, *Loch TP. Bacterial Coldwater Disease Updates. Presented virtually at a Michigan Department of Natural Resources Fisheries Division Meeting, 2022.
• Type: Conference Papers and Presentations Status: Other Year Published: 2022 Citation: *Loch TP. MI DNR and MSU-AAHL collaborations: A proven recipe for healthier fishes. Presented virtually at the Michigan Department of Natural Resources Management Team Meeting, 2022.
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Lennox SMG, Shavalier MA, Brenden TO, Knupp CK, Call DR, Soto E, *Loch TP. Enhancing bacterial coldwater disease diagnosis and prevention by elucidating the predominant Flavobacterium psychrophilum serovariants in the USA. Presented at the 9th International Symposium of Aquatic Animal Health, Santiago, Chile, 2022.
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: *Lennox SMG, Shavalier MA, Brenden TO, Knupp CK, Call DR, Soto E, Loch TP. Enhancing bacterial coldwater disease diagnosis and prevention by elucidating the predominant Flavobacterium psychrophilum serovariants in the USA. Presented virtually at the American Fisheries Society  Fish Health Section Summer Seminar Series, 2022. Best Student Presentation Award.
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: *Lennox SL, Shavalier MA, Brenden TO, Knupp CK, Call DR, Soto E, Loch TP. Enhancing Bacterial Coldwater Disease Diagnosis And Prevention By Elucidating The Predominant Flavobacterium psychrophilum Serovariants In The USA. Presented at the 17th Annual Michigan State University Department of Fisheries and Wildlife Graduate Student Research Symposium, East Lansing, MI, 2022. *Best Retrospective Student Paper Award*

Progress 09/01/20 to 08/31/21

Outputs
Target Audience:Our target audiences during the current reporting period included: a) two graduate students (Sean Lennox, Michigan State University, MSU; Samantha Darling, MSU) in PD-Loch's laboratory that received scholarly mentorship and training (and also mentored and trained others) in flavobacteriological, microbiological, and molecular techniques, data analyses, and effective scientific communication; b) undergraduate and graduate students enrolled in PD Loch's Principles of Fish and Wildlife Disease lecture and laboratory courses (Fall semester, 2021; 20 students); c) attendees (e.g., university graduate and undergraduate students, MSU faculty and staff, stakeholders from various regional fisheries management agencies) of the 16th Annual Department of Fisheries and Wildlife Graduate Student Organization Research Symposium; d) members of the Great Lakes Fishery Commission - Great Lakes Fish Health Committee (with members from eight Great Lakes states and two Canadian provinces); e) members of the Northeast Fish Health Committee representing 13 US states plus the District of Columbia; f) upper management for the Michigan Department of Natural Resources Fisheries Division and the Pennsylvania Fish and Boat Commission; g) fishery biologists and technicians employed at multiple aquaculture facilities and state fish hatcheries in the USA; h) fish health professionals and aquatic animal veterinarians); i) members of PD-Loch's laboratory during regularly scheduled laboratory meetings; j) international veterinary students and other attendees (n=>800) of the "Three in One" Visiting Scholar Program (organized by Brawijaya University, East Java, Indonesia) hailing from multiple countries in Southeast Asia and Europe; k) international attendees of the 4th International Conference On One Health (ICOH). Our efforts to deliver science-based knowledge that have thus far resulted from this ongoing USDA-NIFA funded project include the following: a) multiple oral presentations (both in-person and virtual) at professional conferences and meetings that were given by PD-Loch and Mr. Sean Lennox (i.e., the graduate student whose research is funded by this USDA-NIFA project); b) outreach and extension via discussions with and presentations to fishery managers, fishery biologists, and aquaculture facility personnel (e.g., through group meetings at hatcheries and aquaculture facilities, through presentations to Michigan Department of Natural Resources Fisheries Division and Pennsylvania Fish and Boat Commission personnel, through presentations to members of the Great Lakes Fishery Commission - Great Lakes Fish Health Committee and members of the Northeast Fish Health Committee); c) laboratory instruction, training, mentorship, discussions, and experiential learning for multiple graduate students during the current reporting period; d) formal classroom instruction (in person) for Michigan State University graduate and undergraduate students enrolled in PD-Loch's Principles of Fish and Wildlife Disease lecture and laboratory courses; and e) multiple virtual seminars and presentations for veterinary students from numerous universities overseas. Changes/Problems:The continued challenges posed by the COVID-19 pandemic have resulted in some delays from the originally proposed project timeline. However and likely with a no-cost extension, we anticipate the objectives and tasks will be completed. What opportunities for training and professional development has the project provided?During year 1 of this ongoing USDA-NIFA funded study, a multitude of training and professional development opportunities have been (and continue to be) afforded to multiple individuals. Training opportunities have included: a) extensive training and mentorship for two graduate students (e.g., Sean Lennox, MSU; Samantha Darling, MSU) in PD-Loch's laboratory resulting from immersive, hands-on experience with flavobacteriological/microbiological/molecular laboratory experiments, data analyses, and activities for enhancing effective oral and written scientific communication skills. Importantly, Mr. Lennox has also taken on an impressive mentorship role for other students, graduate and undergraduate alike, thereby fostering his own scholarly mentorship skills. Likewise, Sean is currently on a research assistantship partially funded by this USDA-NIFA grant, and thus has learned and received training during several graduate level courses towards fulfillment of his MSc requirements; b) discussions and critical thinking related to pathogen diversity and the development of animal disease prevention and control strategies for undergraduate and graduate students enrolled in PD Loch's Principles of Fish and Wildlife Disease lecture and laboratory courses (Fall semester, 2021; 20 students); c) exercises in experimental design, data interpretation, and data analyses for members of PD-Loch's laboratory as experiments, results, and analyses to date were discussed and critical thinking sought; and d) engagement of graduate students in a series of mock interviews with journalism students as a part of the MSU course "Practice of Fisheries and Wildlife Outreach and Engagement" (FW895) course, which is specifically designed to practice and facilitate conveying research details in an accessible and engaging manner. Although COVID-19 continues to hamper attendance of in-person conferences and workshops, Professional Development opportunities that have resulted from this USDA-NIFA funded project during year 1 included: a) enhanced development of effective written and oral scientific communication skills for Mr. Lennox, who gained invaluable experience while authoring his first professional scientific abstract, assisting in the drafting of this progress report, and presenting his research in lab meetings and at the 16th Annual Graduate Student Organization Research Symposium at MSU; b) participation of graduate students involved in this study in multiple professional virtual seminars and conferences (e.g., the 2021 American Fisheries Society - Fish Health Section Virtual Seminar Series on fish health); c) the substantial laboratory and fish health/husbandry/diagnostic expertise that have been gained by two graduates students as members of the MSU - Aquatic Animal Health Laboratory; and d) growth of involved graduate students as scholarly mentors of the next generation of scientists. How have the results been disseminated to communities of interest?New knowledge and results generated during the initial year of this ongoing USDA-NIFA funded project have already been extensively disseminated to communities of interest through multiple avenues, including via: a) four professional presentations; b) two invited virtual seminars that reached >800 attendees from multiple European and southeast Asian countries; c) two didactic university courses (e.g., one lecture-based, one laboratory-based) that are taught by PD-Loch and attended by undergraduate, pre-veterinary, and graduate student enrollees; d) formal and informal discussions with a diversity of aquaculture and natural resource manager personnel; e) formal and informal discussions with academicians, agriculture and natural resources administrators, fish health professionals, aquatic veterinarians, students, and fish health committee members; f) discussions and presentations in regular lab meetings amongst the PDs research group; g) in-class (e.g., FW 894, FW 895) discussions led by the graduate students involved in this USDA-NIFA funded project; h) personal communications (through email, Zoom, ResearchGate, phone calls, in-person) with fish health professionals, aquatic veterinarians, researchers, and students inquiring about flavobacterial fish diseases, associated research, and advice on the prevention and control of flavobacterial infections in fish; and i) formal and informal virtual and phone discussions with members of USDA-NIFA's North Central Regional Aquaculture Center (NCRAC), including several that led to the development of a collaborative team of researchers and aquaculture producers aiming to mitigate flavobacteria-associated losses in the North Central Region. What do you plan to do during the next reporting period to accomplish the goals?Although some delays resulting from the ongoing pandemic have occurred, we anticipate the proposed objectives and tasks will be satisfactorily completed upon completion of the project.

Impacts
What was accomplished under these goals? Impact statement: Flavobacterium psychrophilum (Fp), causative agent of bacterial coldwater disease (BCWD), is a top disease impediment faced by producers of trout and salmon across the USA. This microbe is readily transmitted from infected fish to fish, but also from infected parents to offspring (i.e., vertical transmission) via reproductive fluid and egg-associated Fp transmission, making BCWD prevention and control difficult. Our other recent USDA-NIFA funded research has uncovered substantial US Fp genetic diversity while revealing certain Fp strains are behind most BCWD outbreaks in the USA. Moreover, we have shown that Fp transmission from parents to offspring is a major source of such outbreaks, a concern given that BCWD vaccines are currently unavailable and that current fish egg disinfection protocols are inconsistent at best. In this context and during year 1 of this ongoing USDA-NIFA funded project, our research team has made substantial progress towards eventually arming aquaculturists/hatchery managers with readily implementable tools to break Fp vertical transmission and reduce BCWD-associated losses. Thus far and for the first time ever, we have identified predominating US-Fp molecular serotypes (i.e. how fish immune system "sees" Fp during infections) by analyzing >320 Fp isolates that were recovered from across the USA over the last several decades. These data are not only invaluable for guiding future vaccine development, but were utilized during year 1 to guide the development of a new loop-mediated isothermal amplification (LAMP) assay that is capable of detecting all currently recognized Fp molecular sero-variants. Importantly, our new Fp LAMP assay is now being optimized for eventual "pondside" use, which will allow aquaculture/hatchery personnel to rapidly and accurately detect Fp in infected eggs/reproductive fluids and guide use of "Fp-free" eggs for rearing. Moreover, an immensely positive impact on the scholarly scientific development of multiple graduate students has resulted from the training, mentorship, and professional development opportunities that were thus far afforded by this USDA-NIFA funded study. Collectively and as research progress continues, the newly generated knowledge, tools, and expertise will certainly improve US-farmed trout and salmon productivity while simultaneously enhancing food security and hatchery-reared fish health. Objective 1- To identify the predominating Fp serotypes associated with economic losses in the US trout and salmon industries. Task 1: Serotyping of US-Fp isolates. Following extraction of genomic DNA, the multiplex PCR assay and schema of Rochat et al. (2017) and Avendaño-Herrera et al. (2020) were used to assign 325 cryo-preserved and previously multilocus sequence typing (MLST) typed US-Fp isolates to an Fp molecular serotype (mtype). Thus far, US-Fp isolates most commonly belonged to mtype 0, followed by 1, 2, and 4 respectively, with only 4 isolates being identified as mtype 3. In addition to identifying the most common Fp mtypes within this large US Fp collection, initial analyses have also revealed that some mtypes show strong associations with host species (e.g., mtype 0 and coho salmon). Such a large-scale analysis of US-Fp molecular serotypes has never before been published and will not only be invaluable basis for future vaccine development, but also provided crucial data to guide the development of the new LAMP assay (Obj 2). Objective 2- To develop and test a rapid assay for screening trout/salmon gametes for all known US-Fp variants that hatchery biologists/farmers can use "pondside." Task 2a: Development of Fp qLAMP primers. Using Primer Explorer Version 5 software, primer development was attempted using Fp sequence data for seven housekeeping genes (e.g. trpB, gyrB, dnaK, fumC, murG, tuf, atpA) for all published US Fp geno/sero-variants. Although the substantial Fp genetic diversity did not allow for primer development for 6/7 genes, two regions within gyrB proved promising. Primers were synthesized for one of these regions, with excellent success. Task 2b: Nucleic acid extraction & optimization for field applications. Multiple "less-conventional" DNA extraction methods that are best suited for in-field/"pondside" application have been thus far assessed. Among these, a boiling extraction method has preliminarily proven to be effective and visually indistinguishable (using our new LAMP assay, below) from commercialextraction kits when performed on pure Fp colonies. Further assessment of extractions methods, including on fish reproductive fluids and others tissue, will begin shortly. Task 2c: Development and optimization of Fp LAMP & qLAMP assay. Following the development of 6 new LAMP primers (notably, the use of 6 primers enhances the sensitivity and specificity of the assay while still allowing for binding of all currently recognized Fp MLST genovariants based on in silico analyses) that target a region of the Fp gyrB gene, a novel Fp LAMP assay protocol was successfully developed, reagents acquired, and LAMP reaction mixtures devised. Excitingly, this new LAMP assay successfully detects all Fp isolates belonging to the 5 Fp serovariants that have been tested to date while yielding negative results for all other Flavobacterium spp. tested thus far. Following this initial success, optimization of multiple LAMP parameters have been performed and although are ongoing, have proven highly successful. For example, a range of reaction temperatures were assessed, with an optimal reaction temperature range having now been identified, and multiple LAMP reaction indicator candidates (e.g., calcein and hydroxynaphthol blue, HNB) have been assessed, revealing that HNB provides optimal differentiation (including with the naked eye) between positive and negative samples. Likewise, preliminary results suggest that inclusion of betaine into the new LAMP reaction enhances the sensitivity of the assay. We are strongly encouraged by the success of this new Fp assay/protocol thus far, and once fully optimized, will serve as the foundation of qLAMP efforts (e.g. Tasks 2d-4). Task 2d: Evaluation of Fp qLAMP on "spiked" gamete samples. To begin following completion of Task 2c. Task 3: Evaluation of qLAMP diagnostic sensitivity and specificity. To begin following completion of Task 2c. Task 4: Field evaluations of Fp qLAMP diagnostic sensitivity and specificity. To begin following completion of Task 2c. Objective 3- To test and optimize a novel iodophor-based approach for salmon/trout egg disinfection that is effective against all US-predominating Fp sero/genotypes. Sub-task 5a: Origin of gametes for experiments. Although experiments under Obj. 3 have yet to formally begin, Mr. Sean Lennox (i.e., the graduate student conducting the bulk of this research) has gained substantial expertise in trout/salmon husbandry, as well as hands on experience caring for and hatching salmon and trout eggs using Heath stacks/flow through systems. These experiences will be invaluable as the egg disinfection experiments under Obj. 3 commence. Sub-task 5b: Fp challenge of eggs and iodophor disinfection experiments. To begin during next reporting period. Task 6: Assessing efficacy of the optimized iodophor egg disinfection method for eliminating Fp in Atlantic salmon and rainbow trout facilities. To begin during next reporting period. Objective 4- To enhance best management practices (BMPs) for preventing/controlling BCWD via modeling of newly derived data. To begin during next reporting period. Objective 5- To disseminate enhanced BMPs and training to perform "pondside" testing to aquaculture/hatchery personnel via workshops and webinars. To occur following completion of Obj.1-4.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Lennox S, Shavalier MA, Brenden TO, Knupp CK, Loch TP. Breaking Vertical Transmission Cycles of Virulent Flavobacterium psychrophilum Variants. Presented at the Virtual 16th Annual Department of Fisheries and Wildlife Graduate Student Organization Research Symposium, 2021.
• Type: Other Status: Other Year Published: 2021 Citation: Loch TP, Knupp CK, Lennox S, Shavalier MA, Ivan L, Brenden TO. Prevention And Control Of Bacterial Coldwater Disease In Salmonid Hatcheries: Perspectives From Michigan. Invited virtual presentation, Northeast Fish Health Committee Meeting, 2021.
• Type: Other Status: Other Year Published: 2021 Citation: Loch TP. Improving Fish Health in Michigan, the Great Lakes Basin, & Beyond. Invited presentation, Partnership for Ecosystem Research and Management Board of Directors Meeting, 2021.
• Type: Other Status: Other Year Published: 2021 Citation: Loch TP, Knupp CK, Lennox S, Shavalier MA, Yamashita C, Ivan L, Brenden TO. Bacterial Coldwater Disease: Prevention, Control, & Epidemiology. Invited virtual presentation, Pennsylvania Fish and Boat Commission  Virtual Annual Hatchery Manager Meeting, 2021.
• Type: Conference Papers and Presentations Status: Other Year Published: 2021 Citation: Loch TP. Improving Fish Health Through Collaborative Research. Invited virtual seminar in Three in One Visiting Scholar Program, Brawijaya University, East Java, Indonesia, 2021.
• Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Loch TP. Improving the Health of Wild, Feral, and Captive Fisheries in the Great Lakes Basin of North America Through Collaborative Research. Invited Plenary Seminar, 4th International Conference On One Health (ICOH), Brawijaya University, East Java, Indonesia, 2021.