Detection of Lactococcus spp. in Environmental Samples and Wild Fish from Four Lakes in Southern California

Taylor Abraham1; Zeinab Yazdi1, Eric Littman1, Khalid Shahin1,2,, Taylor Heckman1, Eva Marie Quijano Carde1, Diem Thu Nguyen1, Mark Adkison3, Tresa Veek3, Kavery Mukkatira3, Christine Richey3, Kevin Kwak3, Ian Gardner4, Timothy J. Welch5; Haitham H. Mohammed6, Cesar Ortega7, Ruben Avendaño-Herrera8, Bill Keleher9, Michael W. Hyatt10, Véronique LePage11, Esteban Soto1

1University of California Davis, CA, 95616; 2National Institute of Oceanography and Fisheries, Suez, Egypt, 3California Department of Fish and Wildlife, Rancho Cordova, CA, 95670; 4University of Prince Edward Island, Charlottetown, PE, Canada; 5National Center for Cool and Coldwater Aquaculture, USDA-ARS, Leetown, WV, 25430; 6Department of Aquatic Animal Medicine and Management, Faculty of Veterinary Medicine, Assiut University, Assiut, Egypt; 7Universidad Autónoma del Estado de México, Toluca, Mexico; 8Universidad Andrés Bello and Centro FONDAP INCAR, Viña del Mar, Chile; 9Kennebec River Biosciences, Richmond, ME 04357; 10New York Aquarium, Brooklyn, NY 11224, USA, 11LePage Aquatic Veterinary Services, Guelph, Canada

ABSTRACT

Piscine lactococcosis is an important emerging disease of salmonids in North America. While historically attributed solely to the gram-positive bacteria L. garvieae, recent investigations have revealed that lactococcosis is also caused by the closely related species L. petauri and L. formosensis. These three species are indistinguishable by conventional diagnostic methods, leading to the misidentification of L. petauri or L. formosensis as L. garvieae. This oversight reflects a current lack of comprehensive research into the disease; much about the transmission, pathogenesis, and underlying genetics remain unclear. To gain a better understanding of the presence of these bacteria in California, 359 fish (of 8 species) and 161 environmental samples (soil and water) were collected from four lakes near two affected fish farms during an outbreak of piscine lactococcosis in California in 2020. Lactococcus garvieae was isolated from brains of two largemouth bass (Micropterus salmoides) in one of the lakes by standard microbiological methods. Additionally, Lactococcus spp. were detected in 14 fish (8 bluegills and 6 largemouth bass) from 3 out of the 4 lakes using a recently developed quantitative PCR (qPCR) assay. Of the collected environmental samples, all 4 lakes tested positive for Lactococcus spp. in the soil samples, while 2 of the 4 lakes tested positive in the water samples through qPCR. The second objective of the study was to compare the virulence of L. petauri, L. garvieae and L. formosensis in rainbow trout (Oncorhynchus mykiss) and largemouth bass. Treatment groups were intracelomically injected with representative isolates of L. petauri (n=17), L. garvieae (n=2), or L. formosensis (n=4) previously recovered from cultured or wild fish in North America and monitored for 14 days post-challenge (dpc). Challenged largemouth bass did not show any signs of infection or morbidity post-injection throughout the challenge period. Rainbow trout infected with L. petauri showed clinical signs within 3 dpc and presented a significantly higher cumulative mortality (62.2%; p<0.0001) at 14 dpc when compared to L. garvieae (0%) and L. formosensis (10%) infected treatments (0%), suggesting that North American isolates of L. petauri are more virulent to rainbow trout than North American isolates of L. garvieae and L. formosensis. The current study demonstrates the potential use of the developed qPCR assay in detection of the agents of piscine lactococcosis in the environment, and its potential use as a tool for piscine lactococcosis surveillance in natural and commercial settings.  Additionally, the study confirms L. petauri as a highly pathogenic emerging pathogen of rainbow trout in the USA.