Identification of Flavobacterium and Flexibacter Species by Species-Specific Polymerase Chain Reaction Primers to the 16S Ribosomal RNA Gene

Abstract

Species-specific polymerase chain reaction (PCR) primers have been developed for the identification of the causative agents of warmwater and marine finrot in fish: Flavobacterium columnare (Flexibacter columnaris) and Flexibacter maritimus. Differences in gene sequence in the bacterial small-subunit (16S) ribosomal RNA (rRNA) were used to design the species-specific PCR primers. The previously reported species-specific PCR primers Psy1 and Psy2 for the identification of Flavobacterium psychrophilum (Flexibacter psychrophila), the causative agent of coldwater finrot, were also used to develop a speciation scheme for all three bacterial finrots. These three primer sets were successful in discriminating among yellow-pigmented bacteria as well as in speciating the three major pathogenic flexibacteria to fish. The primer sets were designed to produce uniquely sized subproducts of 16S rRNA for each species: Flavobacterium psychrophilum (1,100 base pairs, bp), F. columnare (800 bp), and Flexibacter maritimus (400 bp). These primers were shown to correctly speciate field isolates in double-blind experiments (P = 0.01).     

Colorimetric Method of Loop-Mediated Isothermal Amplification with the Pre-Addition of Calcein for Detecting Flavobacterium columnare and its Assessment in Tilapia Farms

Flavobacterium columnare, the causative agent of columnaris disease in fish, affects many economically important freshwater fish species. A colorimetric method of loop-mediated isothermal amplification with the pre-addition of calcein (LAMP–calcein) was developed and used to detect the presence of F. columnare in farmed tilapia (Nile Tilapia Oreochromis niloticus and red tilapia [Nile Tilapia × Mozambique Tilapia O. mossambicus]) and rearing water. The detection method, based on a change in color from orange to green, could be performed within 45 min at 63°C. The method was highly specific, as it had no cross-detections with 14 other bacterial species, including other fish pathogens and two Flavobacterium species. The method has a minimum detection limit of 2.2 × 10² F. columnare CFU; thus, it is about 10 times more sensitive than conventional PCR. With this method, F. columnare was detected in gonad, gill, and blood samples from apparently healthy tilapia broodstock as well as in samples of fertilized eggs, newly hatched fry, and rearing water. The bacteria isolated from the blood were further characterized biochemically and found to be phenotypically identical to F. columnare. The amplified products from the LAMP–calcein method had 97% homology with the DNA sequence of F. columnare.

Received May 21, 2014; accepted August 10, 2014     

Attempts to Control Flexibacter columnaris Epizootics in Pond-Reared Channel Catfish by Vaccination

Abstract

Evaluation of bacterins for the immunization of channel catfish Ictalurus punctatus against Flexibacter columnaris was carried out over 5 years. Groups of 60,000–100,000 pondreared channel catfish, weighing an average of 1.5–4.1 g, were vaccinated each year with formalininactivated Flexibacter columnaris bacterins by immersion. Bacterins were prepared from isolates of the preceding year's epizootic. Equal numbers of nonvaccinated channel catfish reared under similar conditions served as controls. Fish were vaccinated during May each year when water temperatures ranged from 16 to 18°C. Data were recorded daily from early June through midSeptember as water temperatures rose to 24–26°C. Deaths were recorded daily, and the number of hours required for antibiotic treatments were monitored. Analysis of the data indicated a general trend toward a beneficial effect of vaccination, particularly for larger fish. Mortality and hours of antibiotic treatment were significantly higher for most control groups than for vaccinated groups.     

Improved Method for Determining Antibiotic Susceptibility of Flavobacterium columnare Isolates by Broth Microdilution

Abstract

A simple and reproducible microdilution method was developed to test the susceptibility of the bacterium Flavobacterium columnare to antibiotics in vitro. The testing was conducted at 28°C for 44–48 h at two dilutions of Mueller–Hinton broth (DMHB) using a standardized inoculum, a reference isolate of Escherichia coli ATCC25922 as a quality control organism, positive and negative control wells, and standardized custom-made microtiter plates. The E. coli ATCC25922 and F. columnare ATCC23463 (the species type strain) had significantly better growth in DMHB at 1:5 (4 g/L) than at 1:7 (3 g/L). The E. coli ATCC25922 was found to be acceptable as a reference isolate and produced minimum inhibitory concentration (MIC) values similar to those in the range published by the Clinical and Laboratory Standards Institute derived using standard Mueller–Hinton broth. The new method was used to determine the MIC of 23 F. columnare isolates (representing the three genotypes of the species) to enrofloxacin, ampicillin, oxytetracycline, erythromycin, florfenicol, flumequine, ormetoprim/sulfadimethoxine, and oxolinic acid.     

Phenotypic and genetic relationships of so-called Moraxella (Pasteurella) anatipestifer to the Flavobacterium/Cytophaga group

So-called Moraxella (or Pasteurella) anatipestifer and members of the Flavobacterium/Cytophaga group exhibit remarkable common features: lack of flagellation, low guanine + cytosine content of the chromosomal DNA, production of menaquinones and branched-chain fatty acids, absence of carbohydrate fermentation, and similar patterns of hydrolytic enzymes. Using the renaturation method of DNA:DNA hybridization two urease-negative European isolates and the urease-positive type strain (which was isolated in the United States) of M. P. anatipestifer were shown to have about 85% of their genome DNA base sequences in common; they may represent two subspecies. The type strain of this species was neither measurably related to the type species of the genus Moraxella nor to selected members of the family Pasteurellaceae (Pohl 1981). On the other hand, low but significant degrees of DNA binding between selected strains of so-called M. anatipestifer, Cytophaga marinoflava, Flavobacterium meningosepticum, F. odoratum and F. pectinovorum were observed. On the basis of these findings the transfer of the so-called M. anatipestifer to the Flavobacterium/Cytophaga group (family Cytophagaceae) is proposed. More detailed investigations are required to establish its relationship at the genus level.     

Characterization of four Flavobacterium columnare (Flexibacter columnaris) strains isolated from tropical fish

Four Flavobacterium columnare strains (AJS 1–4) were isolated from black mollies (Poecilia sphenops) and platies (Xiphophorus maculatus), showing white spots on the back, head and skin ulcers. The isolates developed characteristic rhizoid yellow pigmented colonies on Shieh agar and typical growth in Shieh broth. They were Gram-negative, filamentous bacteria exhibiting flexing movements. When compared to F. columnare strains isolated from temperate fish, it was noted that the four strains originating from tropical aquarium fish are more capable of growing at higher temperatures, the opposite being true for the strains isolated from temperate fish. Biochemical characterization and agglutination tests proved that the isolated strains could be classified as F. columnare. Low minimal inhibitory concentration (MIC) values were found for chloramphenicol, erythromycin, furazolidone, kanamycin, lincomycin, nalidixic acid, oxytetracycline and streptomycin. MIC values were high for colistin, sulfamethoxazole and neomycin. Pathogenicity studies were performed on black mollies. When these animals were submersed in an infective solution of the F. columnare strains, a marked difference in virulence was noted among the four isolated strains, strain AJS 1 being the most virulent one and strain AJS 4 being of low virulence.     

Phylogenetic analysis of hindgut microbiota in Hokkaido native horses compared to light horses

The analysis of 16S rDNA sequence of bacteria in feces of Hokkaido native horses and light horses were performed to compare the hindgut microbiota between the two breeds. One hundred and four bacterial 16S rDNA clones (57 clones from four native horses and 47 clones from two light horses) were obtained. Only four sequences (3.8% of total sequences) showed 97% or more similarity to known species. The sequences were mainly affiliated with Cytophaga–Flavobacter–Bacteroides and low GC Gram‐positive bacteria (LGCGP). Proportion of LGCGP was higher in light horses. Other phyla including Verrucomicrobia, Spirochaetes and Archaea were detected only for native horses, suggesting high diversity of microbiota in native horses. In LGCGP, clusters related to known cellulolytic species were found only for native horses, while a cluster related to soluble sugar‐utilizing species was detected only for light horses. The library composition‐comparing software LIBSHUFF showed significant (P < 0.05) difference of fecal microbiota between the horse breeds. The number of Fibrobacter succinogenes‐related sequence and the frequency of detection of novel groups were found to be higher in native horses by selective amplification analysis. The results suggest that genetic diversity and population size of the F. succinogenes group are higher in the hindgut of native horses.     

Isolation and Partial Characterization of Extracellular Proteases Produced by Isolates of Flavobacterium columnare Derived from Channel Catfish

Abstract

Proteases of 23 isolates of Flavobacterium columnare derived primarily from channel catfish Ictalurus punctatus raised in the southeastern United States were isolated and partially characterized. The bacterial isolates were divided into two groups according to the apparent molecular masses of proteases after zymographic resolution by nonreducing, nondenaturing sodium dodccyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) with gelatin as the protease substrate. The 15 isolates in group 1 had two proteases with apparent molecular masses of 58 and 53.5 kilodaltons (kDa). Eight group-2 isolates produced three proteases with apparent molecular masses of 59.5, 48, and 44.5 kDa. Culture medium had an effect on the amount of protease produced by F. columnare LA 88–173. More protease was produced in a medium with low nutrients and salt (Ordal's medium) than in media with higher concentrations of nutrients or salts (TYES, Hsu-Shotts, modified Shieh's media). No differences were observed in the apparent molecular masses of the two proteases of F. columnare LA 88–173 produced in the various media or with different incubation times. Two proteases with apparent molecular masses of 58 and 53.5 kDa were seen as early as I d after inoculation, and these molecular masses did not change during the 7-d experiment. A sharp increase in protease production occurred during the first 24 h of incubation with minimal increase during the remaining 7 d of the experiment. All 23 isolates of F. columnare degraded the gelatin and casein incorporated into TYES agar medium but only 7 of the 23 isolates degraded elastin.     

Technique for Identifying Flavobacterium columnare Using Whole-Cell Fatty Acid Profiles

Abstract

Isolates of Flavobacterium columnare (29 from diseased fish and three American Type Culture Collection cultures [ATCC 23463, 49512, 43622]) were identified by use of biochemical characteristics prior to generating whole-cell fatty acid profiles. The microbial identification system (MIS; Microbial ID, Newark, Delaware), a gas chromatography system, was used to generate the fatty acid profiles of F. columnare. The MIS contains databases of clinically and environmentally important bacteria that are represented by over 100 genera, including Flavobacterium spp. (F. aquatile [ATCC 11947] and F. mizutaii). Flavobacterium columnare is not included in the databases because it does not grow on standard media. Fatty acid profiles of F. columnare were generated with the CLIN40 protocol established by MIS after growth of the bacteria in modified Shieh broth. The fatty acid composition of F. columnare isolates determined by the CLIN40 method consisted of 10 major fatty acids (those present at levels > 1%): 11-methyl-dodecanoic acid (13:0 iso [the term “iso” designates the methyl group at the penultimate carbon atom]), 13-methyl tetradecenoic acid isomer G (15:1 iso G), 13-methyl tetradecanoic acid (15:0 iso), 12-methyl tetradecanoic acid (15:0 anteiso [the term “anteiso” designates the methyl group at the third carbon atom from the end]), pentadecanoic acid (15:0), 14-methyl pentadecanoic acid (16:0 iso), 3-hydroxy-13-methyl tetradecanoic acid (15:0 iso 3OH), 15-methyl cis-9-hexadecenoic acid (iso 17:1 ω9c), 3-hydroxy-14-methyl pentadecanoic acid (16:0 iso 3OH), and 3-hydroxy-15-methyl hexadecanoic acid (17:0 iso 3OH) (94.8% of profile). Five fatty acids found in the highest percentages from all isolates (CLIN40 method) included 15:1 iso G (16.12%), 15:0 iso (46.54%), 15:0 iso 3OH (6.81%), iso 17:1 ω9c (7.32%), and 17:0 iso 3OH (9.42%). Fatty acid profiles were also established by means of the MIS rapid protocol (RCLN50) in which identifications can be completed in 7 min instead of 20 min. Fatty acids found in the highest percentages for the RCLN50 protocol included 15:1 iso G (15.36%), 15:0 iso (43.03%), 15:0 iso 3OH (7.43%), iso 17:1 ω9c (6.83%), and 17:0 iso 3OH (10.17%). Both methods will allow reliable identification of F. columnare.     

Biofilm Formation on Aquaculture Substrates by Selected Bacterial Fish Pathogens

Abstract

The objective of this study was to determine whether common bacterial catfish pathogens could attach and colonize surfaces commonly found in aquaculture facilities. In addition, we evaluated the role of calcium in biofilm formation. Attachment to polystyrene plates was used to quantify biofilm formation by five bacterial pathogens (i.e., Flavobacterium columnare, Aeromonas hydrophila, Edwardsiella ictaluri, E. tarda, and E. piscicida). Flavobacterium columnare and A. hydrophila formed thick biofilms that were enhanced by calcium supplementation. Biofilm formation was significantly lower in all Edwardsiella species tested and calcium had little to no effect on Edwardsiella biofilm formation. Attachment to natural and artificial surfaces was quantified by a standard plate count method. Scanning electron microscopy (SEM) was used to confirm biofilm formation on the substrates. Flavobacterium columnare formed biofilm on the liner, flexible PVC, and nets. Bamboo prevented F. columnare attachment and inhibited cell growth. Aeromonas hydrophila and E. ictaluri formed biofilm on all materials tested, although significant differences were found among substrates. While E. ictaluri failed to form biofilm on microtiter polystyrene plates, it was able to colonize and multiply on all aquaculture materials tested. Our results demonstrated that common bacterial pathogens had the potential of colonizing surfaces and may use biofilm as reservoirs in fish farms.

Received July 19, 2016; accepted January 19, 2017 Published online April 13, 2017     

Investigation of the Link between Broodstock Infection,Vertical Transmission,and Prevalence of Flavobacterium psychrophilum in Eggs and Progeny of Rainbow Trout and Coho Salmon

Abstract

The etiological agent of bacterial coldwater disease (BCWD), Flavobacterium psychrophilum, can be transmitted both vertically and horizontally. Outbreaks of BCWD can result in significant losses in salmonid aquaculture. Reduction of outbreaks in fry may be possible through implementation of a management strategy in which progeny of heavily infected broodstock are culled from the general population. Diagnostic assays to quantify F. psychrophilum concentrations in tissue samples and confirm presence of the bacterium in ovarian fluid have been previously validated. In the current study, these assays were used to screen 60 female Rainbow Trout Oncorhynchus mykiss and 60 female Coho Salmon O. kisutch broodstock at two aquaculture facilities. Eyed eggs from 10 female broodstock (five fish from each facility) exhibiting graded levels of infection were transferred to the University of Idaho and monitored through early life stages for the presence of F. psychrophilum. Female Rainbow Trout broodstock were not positive for F. psychrophilum by enzyme-linked immunosorbent assay (ELISA) and prevalence was low in these progeny. However, ELISA optical density values for kidney correlated to F. psychrophilum prevalence in progeny (r = 0.938, P < 0.05) of Coho Salmon. Nested PCR on ovarian fluid was not a reliable indicator of vertical transmission in either species as broodstock ovarian fluid results did not correlate to F. psychrophilum prevalence in eyed eggs. Further research with these assays is necessary; however, results from this study indicate that broodstock screening may be a potential tool for evaluating F. psychrophilum infection levels, which could become an important component for disease management.

Received December 19, 2012; accepted December 8, 2013     

Spleen Index and Mannose-Binding Lectin Levels in Four Channel Catfish Families Exhibiting Different Susceptibilities to Flavobacterium columnare and Edwardsiella ictaluri

Abstract

Edwardsiella ictaluri and Flavobacterium columnare are two bacterial pathogens that affect channel catfish Ictalurus punctatus aquaculture. At the Catfish Genetics Research Unit (U.S. Department of Agriculture, Agricultural Research Service), some progress has been made in selectively breeding for resistance to E. ictaluri; however, the susceptibility of these families to F. columnare is not known. Our objectives were to obtain baseline information on the susceptibility of channel catfish families (maintained as part of the selective breeding program) to E. ictaluri and F. columnare and to determine whether the spleen index and plasma levels of mannose-binding lectin (MBL) are predictive indicators of susceptibility to these pathogens. Four channel catfish families were used: family A was randomly chosen from spawns of fish that were not selectively bred for resistance; families B, C, and D were obtained after selection for resistance to E. ictaluri. All four families were immersion challenged with both bacterial pathogens; the spleen index and plasma MBL levels of unchallenged fish from each family were determined. Mean cumulative percent mortality (CPM) after E. ictaluri challenge ranged from 4% to 33% among families. Families A and B were more susceptible to F. columnare (mean CPM of three independent challenges = 95% and 93%) than families C and D (45% and 48%), demonstrating that there is genetic variation in resistance to F. columnare. Spleen index values and MBL levels were not significantly different, indicating that these metrics are not predictive indicators of F. columnare or E. ictaluri susceptibility in the four tested families. Interestingly, the two families that exhibited the highest CPM after F. columnare challenges had the lowest CPM after E. ictaluri challenge. Further research on larger numbers of families is needed to determine whether there is any genetic correlation between resistance to E. ictaluri and resistance to F. columnare.

Received November 18, 2011; accepted February 23, 2012     

Epizootiological Study of Bacterial Cold-Water Disease in Pacific Salmon and Further Characterization of the Etiologic Agent,Flexibacter psychrophila

Abstract

Isolates of Flexibacter psychrophila were obtained from chinook salmon Oncorhynchus tshawytscha and coho salmon O. kisutch that had previously sustained epizootics of coldwater disease. The pathogen was readily isolated from kidney and mucus of convalescent fish. The organisms were relatively inert in most standard microbiological media but were structurally and serologically homogenous by examination of whole cell protein lysates by sodium dodecyl sulfate polyacrylamide gel electrophoresis. In contrast to the homogeneity observed in phenotypic and serologic assays, the isolates studied elaborated varied ribotypes. All isolates produced a single rDNA spacer amplification product of about 240 base pairs.     

Toxicity of Copper Sulfate to Flavobacterium psychrophilum and Rainbow Trout Eggs

Abstract

Tests were conducted to determine the concentrations of copper sulfate needed to kill Flavobacterium psychrophilum, the cause of bacterial coldwater disease, either in vitro or on Rainbow Trout Oncorhynchus mykiss eggs. For the in vitro test, a plastic strip dipped in a solution of F. psychrophilum was exposed for 15 min to copper sulfate solutions of 0, 1, 5, 10, 20, 35, 50, 75, or 100 mg/L. Bacteria were “too numerous to count” at concentrations ≤10 mg/L CuSO₄; significant reductions in prevalence relative to untreated controls were noted for concentrations ≥35 mg/L. However, CFUs were still observed at 50 and 75 mg/L (20% of plates with tryptone yeast extract salts media). No yellow-pigmented CFUs typical of F. psychrophilum were observed at 100 mg/L CuSO₄. For the in vivo test, eggs were exposed for 15 min to 100, 300, 500, and 700 mg/L CuSO₄ or 100 mg/L iodine (control). Survival to hatch was significantly lower at 500 (44.3 ± 15.2%, mean ± SD) or 700 mg/L CuSO₄ (1.7 ± 0.8%) than for controls treated with 100 mg/L iodine (93.6 ± 0.9%) or at copper sulfate concentrations ≤300 mg/L. The 15-min LD₅₀ and LD₁₀ for copper sulfate were 461 mg/L (95% confidence interval: 457–466 mg/L) and 259 mg/L (251–266 mg/L). The prevalence of yellow CFUs at 100 mg/L CuSO₄ (40.0%) was significantly higher than in untreated controls. Significant reductions in yellow CFUs were achieved using 300, 500, or 700 mg/L CuSO₄ (7.5, 2.5, or 0.0% of plates with CFUs, respectively) or 100 mg/L iodine (2.5%), relative to untreated control eggs. Overall, since the concentrations of copper sulfate required to eliminate F. psychrophilum were toxic to the eggs, copper sulfate is not recommended for coldwater disease control in Rainbow Trout eggs based on conditions and parameters in this study.

Received July 7, 2011; accepted March 17, 2013     

Behavior of Rocinela angustata (Isopoda,Aegidae), an Ectoparasite of Alaskan Marine Fishes

Abstract

Observations were made on the feeding and host selection behavior of the aegid isopod Rocinela angustata. Host susceptibility was assessed by placing isopods into laboratory tanks containing 14 common species of Alaska marine fishes. Five species of Sebastes and Blepsias bilobus were attacked within a few minutes of the introduction. Five species—Enophrys bison, Eumicrotremus orbis, Hexagrammos lagocephalus, Pleuronectes asper, and Pholis laeta—were attacked 10–24 h after introduction of the isopods. Three species—Podothecus acipenserinus, Liparis gibbus, and Lycodes brevipes—were not successfully attacked, even when held in isolation with an isopod for 14 d. Starved isopods isolated with B. bilobus attached to fish within 1 h and remained attached 4–14 d. The isopods reattached 1–4 d after detachment. Isopod digestive tracts cleared within 24 h after detachment.     

The Effect of High Total Ammonia Concentration on the Survival of Channel Catfish Experimentally Infected with Flavobacterium columnare

Abstract

Ammonia concentrations in water can affect the severity of Flavobacterium columnare infections in fish. Two trials lasting 7 d each were conducted to determine the effect of a single immersion flush treatment of total ammonia nitrogen (TAN; 15 mg/L) on the survival of channel catfish Ictalurus punctatus infected with F. columnare; the chemical was added while the water flowed continuously through the tanks. Both trials consisted of four treatments: (1) no ammonia exposure and no bacterial challenge (control), (2) ammonia exposure only, (3) bacterial challenge only, and (4) both ammonia exposure and bacterial challenge. Two hours after exposure to ammonia, the highest un-ionized ammonia level was 0.43 mg/L. The percent un-ionized ammonia is based on TAN, temperature, and pH. Caudal fins from three fish in each treatment were sampled at 24 h posttreatment to be analyzed by quantitative real-time polymerase chain reaction (qPCR). No significant difference in survival (mean ± SE) was noted between the channel catfish in treatment 1 (95.2 ± 1.2%) and those in treatment 2 (95.6 ± 1.0%); however, survival in both treatments 1 and 2 differed significantly from that in treatments 3 (8.5 ± 4.5%) and 4 (41.8 ± 12.7%). Treatment 4 catfish had significantly higher survival than treatment 3 catfish. Quantitative PCR data showed that treatment 4 fish had significantly less F. columnare (7.6 × 10⁵) than did treatment 3 fish (1.2 × 10⁷), and treatment 2 fish (8.5 × 10³) had significantly less bacteria than did treatment 1 fish (6.9 × 10⁴), indicating that ammonia limited the F. columnare infection. The highest mean concentration of the bacteria (3.9 × 10⁷) was found on moribund fish. The ammonia concentrations tested did not negatively influence fish survival but interfered with the infection process. An in vitro assay was also conducted to evaluate the direct effects of ammonia on F. columnare.

Received September 15, 2010; accepted May 7, 2011     

Assay to Evaluate the Reaction Kinetics of Chondroitin AC Lyase Produced by Cytophaga columnaris

Abstract

The development of an assay to quantitate chondroitin AC lyase activity of Cytophaga columnaris isolates is described. Assay conditions were defined by using C. columnaris originally isolated from channel catfish Ictalurus punctatus, coho salmon Oncorhynchus kisutch, goldfish Carassius auratus, and striped bass Morone saxatilis affected with clinical columnaris disease. Supernatant and cellular components of broth cultures exhibited strong activity, and rates of chondroitin sulfate degradation ranged from 20.0 to 81.4 μg/(mL.h) for the cell component and from 35.4 to 83.4 μg/(mL.h) for the supernatant. Degradation rates were calculated by simple linear regression analysis, and most correlations ranged from ?0.90 to ?1.00. The assay provided results within 2–3 h, and the enzyme is active under a wide range of pH (5–9) and temperature (10–50°C) conditions. The assay can be used as a simple diagnostic aid to differentiate C. columnaris from Cytophaga psychrophila, but more importantly, as a quantitative tool to explore any relationship between specific chondroitin lyase activity and columnaris disease.     

Flavobacterium psychrophilum Infections in Salmonid Broodstock and Hatchery-Propagated Stocks of the Great Lakes Basin

Bacterial coldwater disease (BCWD), caused by Flavobacterium psychrophilum, threatens wild and propagated salmonids worldwide and leads to substantial economic losses. In addition to being horizontally transmitted, F. psychrophilum can be passed from infected parents to their progeny, furthering the negative impacts of this pathogen. In Michigan, both feral and captive salmonid broodstocks are the gamete sources used in fishery propagation efforts. A 5-year study was initiated to follow the prevalence of systemic F. psychrophilum infections in feral broodstocks of four species (steelhead Oncorhynchus mykiss [potadromous Rainbow Trout]; Coho Salmon O. kisutch; Chinook Salmon O. tshawytscha; and Atlantic Salmon Salmo salar) residing in three Great Lakes watersheds. Additionally, captive broodstocks of four species (Rainbow Trout, Brown Trout Salmo trutta, Lake Trout Salvelinus namaycush, and Brook Trout Salvelinus fontinalis) maintained at two facilities were assessed for the presence of F. psychrophilum. The resultant offspring from each broodstock population were sampled for F. psychrophilum infections multiple times throughout hatchery residency. Using selective flavobacterial culture and PCR confirmation, F. psychrophilum was detected in all broodstocks except the captive Lake Trout and Brook Trout. Logistic regression analysis demonstrated that among the infected feral broodstocks, Chinook Salmon from the Lake Michigan watershed had the highest prevalence of systemic F. psychrophilum infection (mean = 63.2%). Among the captive broodstocks, the Gilchrist Creek strain of Brown Trout had the highest infection prevalence (mean = 5%). Collectively, the captive broodstocks were found to have significantly lower infection prevalence than the feral broodstocks. Despite the high prevalence of systemic F. psychrophilum infections in many broodstock populations, the bacterium was rarely detected in their progeny during hatchery rearing. However, heavy losses associated with clinical BCWD outbreaks did occur. Collectively, our results reinforce that BCWD continues to threaten Great Lakes basin salmonids.

Received April 6, 2015; accepted August 25, 2015     

Guide for Authors

Abstract

The etiological agent of bacterial cold-water disease, Flavobacterium psychrophilum, can cause significant losses of salmonid fishes in aquaculture facilities. Few studies describing the value of media components on the growth of F. psychrophilum are available in the literature. We therefore conducted a study that began with the standard enriched Anacker-Ordal broth (EAO) and over the course of multiple iterations evaluated the effects of various media supplements by adding or subtracting them from the base EAO medium. Different media formulations were made, and samples were removed from each broth formulation every 24 h for 72 h. From those samples we determined bacterial density by measuring absorbance values with a spectrophotometer. The medium with the highest absorbance value from one iteration was used as the base medium in the next iteration. Using this iterative approach, we determined that sodium acetate, calcium chloride, and magnesium sulfate inhibit growth and that maltose has no effect on the proliferation of the bacterium. The addition of skimmed milk (0.2%) and horse serum (1%) appears to provide a slight improvement in bacterial proliferation. Variations in agar concentration had no effect on the growth of the bacterium. Even though the addition and removal of some ingredients increased the mean absorbance values, the benefit of these substitutions was not significant. Even so, we found that the growth of F. psychrophilum in EAO was better than that in two other widely used media: tryptone-yeast extract salts and maltose infused tryptone-yeast extract salts.

Received August 1, 2011; accepted December 6, 2011     

Development of Similar Broth Microdilution Methods to Determine the Antimicrobial Susceptibility of Flavobacterium columnare and F. psychrophilum

Flavobacterium columnare and F. psychrophilum are major fish pathogens that cause diseases that may require antimicrobial therapy. Choice of appropriate treatment is dependent upon determining the antimicrobial susceptibility of isolates. Therefore we optimized methods for broth microdilution testing of F. columnare and F. psychrophilum to facilitate standardizing an antimicrobial susceptibility test. We developed adaptations to make reproducible broth inoculums and confirmed the proper incubation time and media composition. We tested the stability of potential quality-control bacteria and compared test results between different operators. Log phase occurred at 48 h for F. columnare and 72–96 h for F. psychrophilum, confirming the test should be incubated at 28°C for approximately 48 h and at 18°C for approximately 96 h, respectively. The most consistent susceptibility results were achieved with plain, 4-g/L, dilute Mueller–Hinton broth supplemented with dilute calcium and magnesium. Supplementing the broth with horse serum did not improve growth. The quality-control strains, Escherichia coli ATCC 25922 and Aeromonas salmonicida subsp. salmonicida ATCC 33658, yielded stable minimal inhibitory concentrations (MIC) against all seven antimicrobials tested after 30 passes at 28°C and 15 passes at 18°C. In comparison tests, most MICs of the isolates agreed 100% within one drug dilution for ampicillin, florfenicol, and oxytetracycline. The agreement was lower with the ormetoprim–sulfdimethoxine combination, but there was at least 75% agreement for all but one isolate. These experiments have provided methods to help standardize antimicrobial susceptibility testing of these nutritionally fastidious aquatic bacteria.

Received June 24, 2015; accepted October 2, 2015.