Characterization of trout granulomatous virus (TGV) infection in rainbow trout gills reveals hallmarks of immune evasion and endothelial system dysfunction

Ximena Amortegui1*, Sharon Karniely2, Avi Eldar2, Eran Bacharach3, Irene Salinas1

1University of New Mexico, Department of Biology, Center for Evolution and Theoretical Immunology, Albuquerque, NM87131; 2Department of Virology, Kimron Veterinary Institute, Bet Dagan 50250, Israel; 3 The Shmunis School of Biomedicine and Cancer Research, The George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel-Aviv 69012, Israel.

Economic losses associated with disease outbreaks threaten the sustainability of the fish farming industry. Recently, we discovered a new nidovirus infecting farmed rainbow trout (Oncorhynchus mykiss) in Northern Israel named Trout Granulomatous Virus (TGV). While TGV causes characteristic granuloma lesions in the liver, it can also infect other organs, such as the gills, heart, spleen and gut. Interestingly, trout infected with TGV experience anemia, as evidenced by the gill pallor during necropsy procedures. However, the specific impacts of TGV on the trout gill including which cells are infected and what immune responses are induced are currently unknown.

The goal of this study is to evaluate and characterize the histopathological damage, as well as the immune response in gills of TGV infected rainbow trout during a natural outbreak. We sampled eight infected fish from a rainbow trout farm in Israel with current natural TGV outbreak. We used three control uninfected rainbow trout from the Salinas laboratory at the University of New Mexico as baseline controls. Histopathological scoring of TGV infected fish revealed a diversity of pathologies including hemorrhagic lesions, edema/epithelial lifting, lamellar fusion, infiltration of eosinophilic granular cells (EGCs), cell debris and goblet cell hyperplasia. Interestingly, pathology scores were poorly correlated with TGV viral copies in the gills, conducted by RT-qPCR. Interestingly, we identified granulomas in the gill of one infected fish.

Immunofluorescence staining with a newly developed anti-TGV N protein antibody revealed presence of viral infected cells in the secondary lamella, particularly at the edges. Additionally, immune cells of myeloid characteristics were highly positive for TGV N, and were found in areas of lamellar fusion, near the cartilage, inside gill granulomas and the ILT. MHC-II antibody staining revealed 1) Presence of myeloid cells infiltrating the cartilage of the primary lamella 2) Expression of MHC-II in putative endothelial cells at the base of the secondary lamellae, where large edema pockets were identified. Furthermore, IgM staining revealed presence of secreted IgM pockets/pools in the edema regions and this IgM was not associated with any cell.

Combined, our results indicate that the gills are a primary target of TGV which causes drastic hemorrhagic, epithelial and endothelial local gill damage along with strong infiltrations of immune cells. The presence of infected myeloid cells in the gills suggests that these cells act as a mechanism for viral dissemination across different organs and likely contribute to viral immune evasion via granuloma formation.

Evaluating Oral And Immersion Delivery Of An Edwardsiella piscicida Vaccine In Hybrid Catfish (Ictalurus punctatus × Ictalurus furcatus)

1Sujita Balami, 1Cynthia C. Ware, 2David J. Wise, 3Larry A. Hanson, 4Bradley M. Richardson, 2,5Taylor Heckman, 6Benjamin LaFrentz, 1Divya Rose, 7Esteban Soto, and 1Matt J. Griffin

1Department of Pathobiology and Population Medicine, College of Veterinary Medicine, Mississippi State University, Stoneville, MS, 38776; 2Mississippi Agriculture and Forestry Experiment Station, Delta Research and Extension Center, Mississippi State University, Stoneville, MS, 38776; 3Department of Comparative Biomedical Sciences, College of Veterinary Medicine, Mississippi State University, Mississippi State, MS, 39762; 4Warmwater Aquaculture Research Unit, United States Department of Agriculture – Agricultural Research Service, Stoneville, MS, 38776; 5Department of Wildlife, Fisheries and Aquaculture, Mississippi State University, Stoneville, MS, 38776; 6Aquatic Animal Health Research Unit, United States Department of Agriculture – Agricultural Research Service (USDA-ARS), Auburn, Alabama, 36832 ; 7Department of Medicine & Epidemiology, School of Veterinary Medicine, University of California, Davis, 95616

Edwardsiella piscicida is a gram-negative pathogen that negatively impacts catfish aquaculture. Channel (Ictalurus punctatus) × blue catfish (Ictalurus furcatus) hybrids are widely cultured in the US due to several favorable traits, including improved growth and feed conversion, greater tolerance to low dissolved oxygen, and reduced susceptibility to disease. Increased hybrid production has coincided with the emergence of E. piscicida-associated edwardsiellosis. Hybrid catfish represent approximately 50% of diagnostic submissions to the Mississippi State University College of Veterinary Medicine’s Aquatic Research and Diagnostic Laboratory in Stoneville, MS, but account for over 90% of E. piscicida diagnoses. Despite this, there are no commercially available vaccines for E. piscicida in the US.  In the current study, 17 strains of E. piscicida from five discrete phyletic lineages were attenuated by successive passage on plates containing increasing concentrations of rifampicin (RIF), to a final concentration of 360 µg/mL RIF. Attenuation was evaluated using intracoelomic injections in hybrid catfish, and four candidates were identified as both attenuated and protective against subsequent rechallenge with wild-type E. piscicida S11-285. These candidates were evaluated as both oral and immersion vaccines. Orally vaccinated fish were fed a one-time dose of vaccine-coated feed (~4×10⁷ CFU/g of feed), while immersion-vaccinated fish were immunized via a 1-hour immersion bath in ~1×10⁷ CFU/mL.  Forty-two days post-vaccination (dpv), fish were challenged with wild-type E. piscicida strain S11-285 (~4×10⁶ CFU/fish). One fish per tank was sampled at 1, 2, and 42 dpv to evaluate bacterial persistence in the kidney, and serum was harvested to assess anti-E. piscicida IgM activity. No mortality was observed in any treatment group following vaccination, indicating a high level of safety. All candidates were successfully reisolated from orally vaccinated fish at 1 dpv, indicating the attenuated strains can infect fish in the absence of clinical disease. In contrast, only two candidates were recovered from immersion-vaccinated fish at this time point. At 2 dpv, three candidates were recovered from the kidneys of orally vaccinated fish, while only one candidate was isolated from immersion-vaccinated fish. No bacteria were isolated from kidneys at 42 dpv in any treatment, indicating clearance of all vaccine isolates.  Unfortunately, the challenge dose delivered was extreme, resulting in >80% mortality across all treatment groups. Still, two isolates demonstrated improved survival compared to the control and other vaccine treatments. Furthermore, anti-E. piscicida IgM activity in these treatments was elevated. These findings suggest these candidates have potential as oral and immersion vaccines for hybrid catfish against E. piscicida-associated edwardsiellosis and warrant further investigation for efficacy at less extreme challenge doses.

Pronounced Temporal Shifts Define Pond Microbiome Dynamics In Catfish Production Ponds

1,2Divya Rose, 3Caitlin E. Older, 3Bradley M. Richardson, 1,2Sujita Balami, 1,2Lester H. Khoo, 1,2Ethan Woodyard, 4Jonah Nguyen, 4Larry A. Hanson, 5Andy Perkins, 4T. Graham Rosser and 1,2Matt J. Griffin

1Department of Pathobiology and Population Medicine, College of Veterinary Medicine, Mississippi State University, Stoneville, MS 38776; 2Thad Cochran National Warmwater Aquaculture Center, Delta Research and Extension Center, Mississippi State University, Stoneville, MS 38776; 3Warmwater Aquaculture Research Unit, Agricultural Research Service, U.S. Department of Agriculture, Stoneville, MS 38776; 4Department of Comparative Biomedical Sciences, College of Veterinary Medicine, Mississippi State University, Mississippi State, MS 39762; 5Department of Computer Science and Engineering, Mississippi State University, MS.

Advancements in high-throughput sequencing technologies have revolutionized environmental microbiome research, enabling broad applications across various biological disciplines. Amplicon-based sequencing, particularly targeting conserved marker genes such as 16S rRNA, has facilitated comprehensive profiling of microbial communities in diverse environments. This has provided critical insights into microbial community dynamics and supported the development of intervention strategies during dysbiosis. In U.S. aquaculture, the catfish industry represents the largest finfish sector and has seen an increase in microbiome research. However, most studies have predominantly focused on host-associated microbiomes, with comparatively less research focusing on environmental microbiomes such as those of pond water. Unlike terrestrial animals, fish are in constant interaction with their surrounding aquatic environment, which serves as a reservoir for a diverse array of microbial taxa. Characterizing these microbial communities is essential for understanding ecosystem function and health management in aquaculture. In the present study, water samples from 14 earthen fingerling production ponds were collected at nine different time points over a production season to monitor microbial community dynamics and compare between two different antibiotic treatments (Florfenicol and sulfadimethoxine: ormetoprim; 1:5). Previous research has demonstrated that pond water microbiomes can vary significantly between farms, even when key variables such as host species, feed, climate, and treatment protocols are largely constant. These findings show the strong influence of farm-specific management practices on microbial community structure, complicating between-farm comparisons. In light of these findings, the current study focused on a single commercial aquaculture facility producing channel catfish fingerlings. Microbial communities were characterized using short-read sequencing of the 16S rRNA gene (V4 region). Data were processed using QIIME2. Longitudinal changes in taxa abundance were analyzed using Linear Models for Differential Abundance (LinDA) in R. First-differences and first-distances were calculated from alpha and beta diversity results, respectively, to assess temporal changes in diversity. Results suggest time was the strongest driver of microbial community shifts. Although temperature and day length were not directly measured, both vary predictably with time in outdoor pond systems. As such, the observed temporal patterns likely reflect the combined influence of seasonal changes in temperature, photoperiod, and production phase, all of which contribute to shape microbial communities in catfish ponds. This study highlights the highly dynamic and fluctuating nature of microbial communities in open, earthen catfish production systems. Additionally, the findings suggest the influence of medicated feeds on the pond environment may be transient or are masked by environmental and temporal factors that more strongly influence the microbial community dynamics over the course of a production season.