Bacterial Numbers from Landlocked Fall Chinook Salmon Eyed Eggs Subjected to Various Formalin Treatments as Determined by Scanning Electron Microscopy and Bacteriological Culture Methods

Abstract

This study compared two methods of enumerating bacteria adhered to the external membrane of eggs of landlocked fall Chinook salmon Oncorhynchus tshawytscha that were subjected to different formalin treatment regimes from egg eye‐up to fry hatch. Bacterial numbers were recorded by either directly counting bacteria via a scanning electron microscope (SEM) or via established bacterial culture methods that provided the number of colony‐forming units. Treatment regimes consisted of a daily 15‐min exposure to formalin at either 500 mg/L or 1,667 mg/ L or no formalin initially followed by exposure to a 1,667‐mg/L dose starting 7 d after eye‐up. A control group did not receive any formalin treatments throughout the experiment. No significant correlation was observed between the number of colony‐forming units (CFUs) per square millimeter of egg membrane determined by plate culture and the number of bacteria per square millimeter counted with the SEM. Bacterial numbers determined by the SEM were 40–120 times greater than those obtained by plate culture. The SEM numbers were correlated to embryo survival, but CFU numbers were not. For both counting methods, bacterial numbers among the untreated controls increased over time. Of the three species of bacteria identified in cultures (Flavobacterium columnare, Sphingobacterium spp., and Pseudomonas spp.), F. columnare was predominant. Embryo survival in the trays that did not receive any formalin was significantly less than that in all of the other treatments, but there was no significant difference in survival among any of the three formalin treatment regimes.

Characterization of Bacteria Isolated from Landlocked Fall Chinook Salmon Eggs from Lake Oahe, South Dakota

“…Neither is the isolation of Pseudomonas, another very common freshwater bacterial group (Allen et al 1983), which has been identified previously on stream-incubated salmonid eggs (Bell et al 1971). Unlike the results of Barnes et al (2005), the Flavobacterium on the 27-d-old eggs, although capable of hydrolyzing casein, were not F. columnare, which can be easily misidentified using molecular techniques (Darwish et al 2004), and may represent an undescribed species (the closest GenBank matches exhibited only 97% identity, often considered as the level of identity representing separate bacterial species). Flavobacterium johnsoniae is pathogenic to cultured fish, producing skin lesions similar to those made by F. columnare (Carson et al 1993;Soltani et al 1994).…”

“…Plates were incubated for 6 d, and 30 randomly selected bacterial colonies were transferred to slants of R2A agar and characterized according to colony morphology, gram-staining (Koneman et al 1997), cell morphology, the presence of catalase, presence of cytochrome oxidase, growth and fermentation of glucose in Hugh and Leifson's oxidationfermentation media, hydrolysis of casein in milk agar, hydrolysis of starch, and growth, motility, H 2 S production, and indole production in sulfide-indole-motility (SIM) media (Collins et al 1995;MacFaddin 2000). After removal of the initial egg samples from the entire batch of eggs, the remaining eggs were placed in vertical-flow incubators (Marisource, Fife, Washington) and reared as described by Barnes et al (2005), receiving daily, 15-min, antifungal treatments of 1,667 mg/L formalin. After 27 d, three samples of 10 eggs each were removed and processed as previously described to remove surface bacteria and were subsequently plated on R2A and Cytophaga agar.…”

“…Although this mortality is probably due to a number of factors, bacteria isolated during egg incubation have been implicated in these deaths in several studies (Barnes et al 1997(Barnes et al , 2005Stephenson et al 2003). Other researchers have also suggested that bacteria may negatively impact egg survival (Sauter et al 1987;Barker et al 1989;Holcomb et al 2005), although this relationship is far from certain (Barker et al 1990;Omnes et al 1993).…”

“…Flavobacterium columnare, a bacterium that secretes extracellular proteases and is pathogenic to fish, was isolated from eggs of Lake Oahe Chinook Salmon by Barnes et al (2005), suggesting that this species may be implicated in egg mortal-*Corresponding author: mike.barnes@state.sd.us ity. However, the experimental inoculation of Rainbow Trout O. mykiss eggs with F. columnare did not result in increased egg mortality (Barnes et al 2009).…”

Gamete-associated flavobacteria of the oviparous Chinook salmon (Oncorhynchus tshawytscha) in lakes Michigan and Huron, North America

“…Ten flavobacterial taxa were recovered from the eggs and ovarian fluids of feral female Chinook salmon examined in this study, only one of which (e.g., F. psychrophilum) is wellknown for its penchant for infecting salmonid eggs and has often been found in the ovarian fluid of infected fish (Brown et al, 1997;Cipriano, 2015), whereas one other (e.g., F. columnare) is recognized as readily able to colonize salmonid eggs (Barnes et al, 2005). However, we are unaware of any other studies documenting such a diversity of flavobacteria associated with salmonid eggs and reproductive fluids that originated from the body cavity (i.e., not yet exposed to the external aquatic environment).…”