Intraspecific Variability of Edwardsiella piscicida Recovered from Farm-raised Catfish and Cross-protective Efficacy of a Live-attenuated Edwardsiella ictaluri Vaccine

 

 Adrián López-Porras1, Matt J. Griffin2, David J. Wise1, Thomas G. Rosser3, Suja Aarattuthodiyil1

1Mississippi Agriculture and Forestry Experiment Station, Thad Cochran National Warmwater Aquaculture Center, Delta Research and Extension Center, Mississippi State University, Stoneville, MS 38776; 2Aquatic Research and Diagnostic Laboratory, College of Veterinary Medicine, Thad Cochran National Warmwater Aquaculture Center, Delta Research and Extension Center, Mississippi State University, Stoneville, MS 38776; 3Department of Basic Sciences, College of Veterinary Medicine, Mississippi State University, Mississippi State, MS 39762

ABSTRACT

Edwardsiella piscicida is a Gram-negative enteric pathogen associated with significant losses in global temperate and warmwater aquaculture. Recent work has identified an emergence of E. piscicida in farm-raised channel (Ictalurus punctatus) ´ blue (I. furcatus) hybrid catfish in the southeastern United States. A survey of 3,000+ diagnostic submissions (2013-2017) to the Aquatic Research and Diagnostic Laboratory (ARDL) at the National Warmwater Aquaculture Center in Stoneville, MS, revealed hybrids made up ~40% of diagnostic submissions, yet accounted for >90% of E. piscicida diagnoses. This emergence of E. piscicida in hybrid catfish is worrisome given current industry trends towards increased hybrid production. The objectives of this project were to 1) assess intraspecific genetic variability of E. piscicida isolates recovered from disease outbreaks in cultured channel and hybrid catfish in Mississippi 2) determine virulence associations among genetic variants, and 3) evaluate efficacy of a live, attenuated E. ictaluri vaccine to protect channel and hybrid catfish against heterologous E. piscicida isolates. Repetitive extragenic palindromic sequence-based PCR (Rep-PCR) employing Enterobacterial Repetitive Intergenic Consensus (ERIC) primers was used to analyze 158 archived E. piscicida isolates collected from diagnostic submissions to the ARDL during 2007-2018. Analysis revealed five discrete genetic groups. A subsample of 39 temporally discrete E. piscicida isolates, representing each rep-PCR phylogroup, were chosen for further molecular typing using BOX and GTG5 rep-PCR primers, in addition to virulence gene assessment and multi-locus sequence analysis (MLSA) targeting select housekeeping genes (glucose-6-phosphate isomerase [Pgi]; phosphoglucomutase [Pho]; gyrase B [gyrB]). The MLSA provided greater resolution than rep-PCR, revealing 5 discrete phylogroups which largely correlated with virulence gene profiles. Representatives from each MLSA group were chosen for virulence assessment in channel and hybrid catfish fingerlings. Fish (10-11 g/fish; 20 fish/tank; 5 tanks/treatment group) housed under flow-through conditions (1 L/min at 26-27°C) with supplemental aeration were exposed to E. piscicida variants by intracoelomic injection (~1.5x104 CFU/g of fish). Fish were monitored for morbidity and mortality for 14 days. Cumulative mortality of channel catfish ranged from 22-54% (31.6±6%), while mortality in hybrids ranged from 63-72% (65.8±2%). Across all treatments, mortality was higher in hybrid catfish (p < 0.05). This assessment was followed by investigation of the cross-protective efficacy of a live-attenuated E. ictaluri vaccine against heterologous E. piscicida isolates. Channel and hybrid catfish were immunized by immersion (~1.6x107 CFU/mL) for 1 hr with the E. ictaluri vaccine. Thirty days later, fish were exposed by intracoelomic injection to five discrete E. piscicida variants (~1.5x104 CFU/g of fish) and a wild-type E. ictaluri (~6.6x103 CFU/g of fish). Survival was significantly improved in all vaccinated treatments (p < 0.05). Relative percent survival (RPS) for immunized hybrids exposed to E. piscicida ranged from 54.7- 77.8%, while RPS in channels ranged from 80.5-100%. Similarly, RPS in vaccinated channel and hybrid catfish exposed to wild-type E. ictaluri was 95.8% and 98.1%, respectively. These findings suggest the live-attenuated E. ictaluri vaccine affords significant protection in channel and hybrid catfish against heterologous E. piscicida isolates.