Recovery of Renibacterium salmoninarum from naturally infected salmonine stocks in Michigan using a modified culture protocol

Abstract

Renibacterium salmoninarum, the causative agent of bacterial kidney disease (BKD), is a fastidious and slow-growing bacterium that is extremely difficult to grow in vitro. Herein, we describe a modified primary culture protocol that encompasses a modified bacteriological culture medium and a tissue processing procedure. In order to facilitate the release of R. salmoninarum from granulomatous tissues, kidneys of infected fish were homogenized in a high speed stomacher. The kidney disease medium (KDM2), routinely used for primary culture of R. salmoninarum was modified by the addition of antibiotics and metabolites. When a relatively large inoculum of diluted kidney homogenate was streak-plate inoculated onto the modified KDM2, colonial growth of R. salmoninarum was achieved within 5-7 days, compared to the standard of two weeks or more. The modified procedure was then used to determine the prevalence of R. salmoninarum among representative captive and feral salmonid stocks in Michigan. Prevalence and clinical manifestations varied among species, strains of fish, and locations; however, R. salmoninarum isolates were biochemically homogenous. The improved primary culture procedure described in this study enabled selective and quick isolation of R. salmoninarum. Also, the isolates retrieved in this study constitute a unique biological resource for future studies of R. salmoninarum in the Laurentian Great Lakes.

Resurgence of Salmonid Herpesvirus‐3 Infection (Epizootic Epitheliotropic Disease) in Hatchery‐Propagated Lake Trout in Michigan

“…During their tenure in hatcheries, Lake Trout are susceptible to a number of diseases (Faisal et al. , ), with one of the most lethal being caused by epizootic epitheliotropic disease virus (EEDV; order Herpesvirales, family Alloherpesviridae). This herpesvirus, also known as salmonid herpesvirus‐3, led to the loss of approximately 15 million juvenile, hatchery‐reared Lake Trout in the 1980s.…”

Bacterial infections of Chinook salmon, Oncorhynchus tshawytscha (Walbaum), returning to gamete collecting weirs in Michigan

“…During October 2005, spawning Chinook salmon ( n = 480) were collected, euthanized, and artificially spawned by Michigan Department of Natural Resources (MDNR) personnel from two gamete collecting weirs within the Lake Michigan watershed; the Little Manistee River Weir (LMRW, Manistee County, MI) was sampled four times, and the Medusa Creek Weir (MCW, Charlevoix County, MI) was sampled twice (Table 1). Of the 480 sampled Chinook salmon returning to these weirs, all were tested for the presence of R. salmoninarum using a quantitative enzyme‐linked immunosorbent assay (Q‐ELISA), 420 were sampled for non‐fastidious bacteria on trypticase soy agar (TSA; Remel Inc.) and R. salmoninarum prevalence using the nested polymerase chain reaction (nPCR), while 262 were tested for the presence of R. salmoninarum by culture on modified kidney disease medium (MKDM; Faisal, Eissa & Starliper 2010b; Table 1).…”

“…Upon completion of initial bacteriological sampling, portions of the spleen, as well as portions of the anterior, mid‐ and posterior kidneys were collected aseptically in Whirl Pak ® bags (Nasco) chilled on ice and then processed for both R. salmoninarum isolation according to Faisal, Loch, Brenden, Eissa, Ebener, Wright & Jones (2010a), Faisal et al. (2010b) and for the quantitative enzyme‐linked immunosorbent assay (Pascho & Mulcahy 1987).…”

Clinical Outbreaks of Bacterial Kidney Disease (BKD) in Hatchery-Raised Brook Trout (Salvelinus fontinalis) (Mitchill, 1814): Lessons Learned

“…The homogenized kidney tissues were diluted in 1:4 (w/v) Hank's Balanced Salt Solution (HBSS, Sigma, St. Louis, MO) then stomached for 2 minutes at high-speed in a Biomaster Stomacher-80 (Wolf Laboratories Limited, Pocklington, York, UK) as described in Faisal et al [16].…”

“…Isolation of R. salmoninarum was performed as described by Faisal et al [16]. Briefly, 100 μl of stomached kidney tissue aliquots were spread onto Modified Kidney Disease Medium (MKDM) agar plates modified by Eissa [4] and incubated at 15°C for a period of 10 days.…”