Mississippi State University - Griffin Lab
Mississippi State University - Griffin Lab
Dr. Matt Griffin
Divya Rose1,2, Caitlin E. Older3, Abby Hawkins1, Sujita Balami1,2, Noor-ul-Huda1,2, Cyndi Ware1,2, Todd Byars2, Fernando Yamamoto2,6, Lester H. Khoo1,2, Geoffrey C. Waldbieser3, Hasan Tekedar4, Andy Perkins5, Larry A. Hanson4, T. Graham Rosser4, David J. Wise2,6 and Matt J. Griffin1,2
1Department of Pathobiology and Population Medicine, College of Veterinary Medicine, Mississippi State University, Mississippi State, MS 39762; 2Thad Cochran National Warmwater Aquaculture Center, Delta Research and Extension Center, Mississippi State University, Stoneville, MS 38776; 3Warmwater Aquaculture Research Unit, Agriculture 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; 6Mississippi Agriculture and Forestry Experiment Station, College of Forest Resources, Mississippi State University, MS 38776
Since its inception, catfish aquaculture has undergone significant intensification. While these advancements have significantly improved production and land use efficiency, there has been a concurrent increase of infectious diseases, particularly those of bacterial origin. While an effective vaccine exists for Edwardsiella ictaluri, management of other bacteria remains largely reactive, relying on feed restriction or medicated feeds. The limited availability of approved antibiotics (Aquaflor®, Romet®, Terramycin®) has led to repeated selection pressure, promoting emergence of multidrug-resistant (MDR) bacteria. Research has identified MDR plasmids associated with the catfish pathogens E. ictaluri, E. piscicida, and Plesiomonas shigelloides. Studies have also identified sharing of MDR plasmids among Edwardsiella congeners, highlighting the need to investigate other potential MDR reservoirs and their capacity for plasmid mobilization. Sequence analysis of archived MDR E. ictaluri isolates from the 1990s revealed the presence of MDR plasmids with high sequence similarity to plasmids from contemporary isolates. The primary difference between the historical and more recent isolates is the lack a 4.6 kb gene cassette associated with florfenicol resistance, indicating the extrachromosomal capacity to acquire florfenicol resistance was in place prior to its approval in the late 2000s. Under laboratory conditions, MDR isolates exhibit slower growth rates compared to wild-type strains, indicating a fitness cost to carrying these large (>100 kb) plasmids. Infectivity trials suggest reduced virulence in the MDR strains, further supporting potential reduced fitness of MDR isolates. These analyses also revealed heteroplasmy among the historic E. ictaluri isolates, with observation of multiple 5.6 kb native plasmids. The implications of heteroplasmy remain unclear, although recent studies suggest the pathogen has evolved to maintain only a single copy of these 5.6 kb plasmids. The reservoirs for resistance genes associated with the approved antibiotics (floR, tetD, tetR, sul2) were assessed from water samples from commercial ponds. Pond water was plated (28°C; 48 hr) on non-selective nutrient agar supplemented with florfenicol and oxytetracycline to select for resistant bacteria. Individual colonies were sub-cultured, expanded in broth and sequenced to identify mechanisms of resistance among various host microbes. Lawns from antibiotic plates were harvested and genes related to plasmid mobilization, conjugation, and antibiotic resistance were identified from whole shotgun sequencing using Oxford Nanopore Technologies' adaptive sampling protocol. Analyses confirmed the presence of multiple mobile plasmids carrying resistance genes. Notably, genes facilitating resistance, conjugal transfer and mobilization were present in plasmids of non-pathogenic bacteria, indicating their potential role as reservoirs for spreading resistance through horizontal transfer. This study provides a foundational understanding of antibiotic resistance reservoirs in aquaculture settings and underscores the need for continued research into mitigation strategies for MDR bacteria in aquaculture.
1Sujita Balami, 1Cynthia C. Ware, 2David J. Wise, 3Larry A. Hanson, 4Bradley R. Richardson, 1Noor-Ul-Huda, 1Divya Rose, 5Jing Huang, 5Ashmita Poudel, 6Abby Hawkins, 2Fernando Y. Yamamoto, 7Esteban Soto, and 1Matt J. Griffin
1Department of Pathobiology and Population Medicine, College of Veterinary Medicine, Mississippi State University; 2Mississippi Agriculture and Forestry Experiment Station, Delta Research and Extension Center, Mississippi State University; 3Department of Comparative Biomedical Sciences, College of Veterinary Medicine, Mississippi State University; 4Warmwater Aquaculture Research Unit, United States Department of Agriculture – Agricultural Research Service, Stoneville, MS; 5Department of Wildlife Fisheries and Aquaculture, College of Forest Resources, Mississippi State University; 6College of Veterinary Medicine, Mississippi State University; 7Department of Medicine & Epidemiology, School of Veterinary Medicine, University of California, Davis
In US catfish aquaculture, hybrid catfish have gained popularity as an alternative to Channel Catfish, owing to several favorable production traits, including improved growth and feed conversion, increased tolerance to low dissolved oxygen and reduced susceptibility to several Channel Catfish diseases. However, concurrent with the increased adoption of hybrid catfish has been the emergence of Edwardsiella piscicida in U.S. catfish aquaculture. Hybrid catfish account for >90% of E. piscicida diagnoses at the Aquatic Research and Diagnostic Laboratory in Stoneville, MS, and have caused significant losses in both hybrid and Channel Catfish production systems. A member of the Hafniaceae family, E. piscicida is recognized as a cosmopolitan fish pathogen, with reports from at least 30 different wild and cultured fish species worldwide. Despite its global impact, there are limited commercially viable vaccines for E. piscicida, particularly in catfish. To this end, multiple potential live attenuated vaccine candidates were produced and evaluated for their ability to minimize risk of Edwardsiellosis in catfish. Representative isolates from five discrete E. piscicida phyletic lineages were passed on plates containing increasing concentrations of rifampin (RIF), up to 360 µg/ml. A total of 17 mutant strains were produced and tested for attenuation in two different experimental trials. The first attenuation trial included 10 candidate isolates. Hybrid catfish were concurrently exposed to both the RIF mutant and the wild-type parent through IP injections (~1x104 CFU per g of fish). While low-level mortality (<20%) was observed in all wild-type treatments, mortality was negligible in fish inoculated with rifampin-passed mutants. However, this reduced mortality was significant in only 3 of 10 tested strains. Subsequent rechallenge revealed significant protection against E. piscicida isolate S11-285 for 9 of 10 wild-type strains, but only four of 10 RIF mutants, possibly a result of low immunizing dose or extreme attenuation. The 4 successful mutants yielded relative percent survival of approximately 74-76%. A second trial was conducted similarly, with a higher inoculating dose (~3-6×105 CFU per g of fish) using seven discrete RIF mutants and their wild-type parents. High mortality (>90%) was observed in all wild-type strains, however three RIF mutants showed significant attenuation compared to their wild-type counterparts, with 1.5-3X reductions in mortality. This trial is ongoing and protectivity assessments are pending. The high level of protection conferred by prior exposure to wild-type strains in this and other studies suggests E. piscicida is a viable candidate for vaccination should a suitable attenuated strain be identified.
1,2Noor Ul Huda, 3Brian G. Bosworth, 4Ana Beatriz de S. Farias, 4Fernando Y. Yamamoto, 5Samantha J. Oakey, 1James M. Steadman, 2David P. Marancik and 1Matt J. Griffin
1Thad Cochran National Warmwater Aquaculture Center, Aquatic Research and Diagnostic Laboratory, Department of Pathobiology and Population Medicine, College of Veterinary Medicine, Mississippi State University, Stoneville, MS; 2Department of Pathobiology, St. George’s University, School of Veterinary Medicine, True Blue, Grenada, West Indies; 3Warmwater Aquaculture Research Unit, Agriculture Research Service USDA, Stoneville, MS 38776; 4Thad Cochran National Warmwater Aquaculture Center, Department of Wildlife, Fisheries and Aquaculture, College of Forest Resources, Mississippi State University, Stoneville, MS; 5College of Veterinary Medicine, University of Georgia, Athens, GA
The Delta Select line of Channel Catfish (Ictalurus punctatus) was created in 2006 and has undergone five generations of genetic selection for enhanced growth and improved carcass yield. The first Delta Select were released to U. S. catfish farmers in 2020 and have been widely adopted by producers. While selection efforts targeting enhanced growth and carcass yield continue, scientists are also examining how these selection criteria impact other traits. Catfish producers in the southeastern United States face significant economic losses due to bacterial infections caused by Edwardsiella spp., particularly Edwardsiella ictaluri in Channel Catfish (Ictalurus punctatus) and E. piscicida in Channel x Blue Catfish (I. furcatus) hybrids. Hybrid catfish account for >90% of E. piscicida diagnoses at the Aquatic Research and Diagnostic Laboratory in Stoneville, MS. In experimental trials, the Delta Select Channel Catfish line has shown improved survival in response to E. piscicida infection compared to the Delta Control line, a randomly bred line of Channel Catfish originating from the same base population. The current study was designed to investigate whether this observed resilience in response to E. piscicida infection is transferred to hybrid catfish offsprings produced from the Delta Select Channel Catfish parent. For this purpose, hybrid catfish fingerlings (~38 g) from 14 families of Delta Select and Delta Control lines were arbitrarily distributed to forty, 76-L aquaria (n=28; 2 fish/family) and were challenged with intracoelomic injections of E. piscicida. For each selection line, 8 tanks received a high dose (3.5x 105 CFU/fish) and 8 tanks received a low dose (1.3x 105 CFU/fish) of E. piscicida. For each family, 4 tanks served as unexposed controls. Numerically lower, but statistically non-significant (p>0.05) cumulative mortality was observed in the Delta Select line (25%) compared to the Delta Control line (31%) for the low dose treatment, with comparable mortalities of 38% and 36% in the high dose treatment, respectively. The improved survival of the Delta Select Line of Channel Catfish to E. piscicida infection is a fortuitous benefit of the selection process, as E. piscicida cases in Channel Catfish, while infrequent, can still lead to substantial losses. However, results indicate this improved survival of the Delta Select line of Channel Catfish in response to E. piscicida challenge is not transferred to their hybrid offspring.