Bradley D. Farmer

Basal polarization of the mucosal compartment in Flavobacterium columnare susceptible and resistant channel catfish (Ictalurus punctatus)

The freshwater bacterial pathogen, Flavobacterium columnare, infects a variety of ornamental and farmed fish species worldwide through mucosal attachment points on the gill and skin. While previous studies have demonstrated a chemotactic response of F. columnare to fish mucus, little is known about how host gill mucosal molecular and cellular constituents may impact rates of adhesion, tissue invasion, and ultimately, mortality. Here, we describe the use of RNA-seq to profile gill expression differences between channel catfish (Ictalurus punctatus) differing in their susceptibility to F. columnare both basally (before infection) and at three early timepoints post-infection (1 h, 2 h, and 8 h). After sequencing and de novo assembly of over 350 million 100 base-pair transcript reads, between group comparisons revealed 1714 unique genes differentially expressed greater than 1.5-fold at one or more timepoints. In the large dataset, we focused our analysis on basal differential expression between resistant and susceptible catfish as these genes could potentially reveal genetic and/or environmental factors linked with differential rates of infection. A number of critical innate immune components including iNOS2b, lysozyme C, IL-8, and TNF-alpha were constitutively higher in resistant catfish gill, while susceptible fish showed high expression levels of secreted mucin forms, a rhamnose-binding lectin previously linked to susceptibility, and mucosal immune factors such as CD103 and IL-17. Taken together, the immune and mucin profiles obtained by RNA-seq suggest a basal polarization in the gill mucosa, with susceptible fish possessing a putative mucosecretory, toleragenic phenotype which may predispose them to F. columnare infection.

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 th...

Evaluation of an Ultra-Low-Flow Water Delivery System for Small Experimental Tanks

Abstract An ultra-low-flow water delivery system was developed and tested for use in research studies requiring low flow in small water volumes. Small test systems save on the amount of fish, chemicals, and biologics needed in disease challenge and treatment experiments. The ultra-low-flow system, consisting of a semi-enclosed header tank with a variable-height standpipe and dulled, guarded syringe needle nozzles, can produce flows that result in 1–20 water exchanges/d for a 10-L volume. Water was prefiltered through two inline, 70-μm filters to limit flow loss due to particulates blocking the small-orifice nozzles. Accurate and precise flows are produced by the system, and needle nozzles should last at least 7 d before fouling requires them to be changed; nozzle changes can be made in about 5 s. Flows in the range of 5–135 mL/min were produced by using 3.8-cm-long needles (16, 18, 20, and 21 gauge) with standpipe heights of 15.2, 30.5, and 45.7 cm. Water flows through selected needle nozzles and standpip...

Improved method for determining antibiotic susceptibility of Flavobacterium columnare isolates by broth microdilution.

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 degrees 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.

Peracetic acid is effective for controlling fungus on channel catfish eggs.

Peracetic acid (PAA) is a relatively new compound suggested for use to treat pathogens in aquaculture. It is approved for use in Europe, but not in the United States. This study determined the effectiveness of PAA for fungus control on channel catfish, Ictalurus punctatus (Rafinesque), eggs. The study consisted of five PAA concentrations (2.5, 5, 10, 15 and 20mgL(-1) ) and an untreated control in a flow-through system. A single spawn was used for each replication (N =4). Eggs were treated twice daily until the embryos developed eyes. When hatching was complete for all viable eggs, fry were counted to determine the percent survival in each treatment. Fungal growth was severe in the untreated controls resulting in 11% survival. Treatments of 2.5, 5 and 10mgL(-1) PAA were significantly different from the controls (P<0.05). The highest percent survival of hatched fry was with 5mgL(-1) PAA administered twice daily; the 2.5mgL(-1) PAA treatment had slightly less survival, but gives a higher margin of safety in case of treatment error. Very little fungus was present in treatments receiving 2.5mgL(-1) PAA or higher, and concentrations of 15 and 20mgL(-1) PAA were toxic to the eggs. The mean survivals in the 0, 2.5, 5, 10, 15 and 20mgL(-1) PAA treatments were 11%, 60%, 63%, 62%, 32% and 0%, respectively. Therefore, PAA may be a compound that merits further investigations regarding its use in U.S. aquaculture.

Longevity of Bolbophorus damnificus Infections in Channel Catfish

The digenean Bolbophorus damnificus infects commercial channel catfish Ictalurus punctatus, causing mortality, lower feed consumption, and reduced growth in surviving fish. The purpose of this study was to determine the length of time for which B. damnificus prodiplostomulum metacercariae (juvenile trematode stage that infects fish) would remain viable (parasite appearing to be intact or exhibiting movement) in channel catfish. Fish (n = 210) were infected with molecularly confirmed B. damnificus cercariae harvested from naturally infected marsh rams-horn snails Planorbella trivolvis. During the first sampling (at 20 d postinfection), 8.3 +/- 3.6 metacercariae/fish (mean +/- SD) were found in the host muscle and visceral organs. The channel catfish were then acclimated to a water temperature of either 18 degrees C or 28 degrees C. After 11 months, 6.8 +/- 3.5 and 5.9 +/- 3.0 metacercariae/fish were found in groups held at 18 degrees C and 28 degrees C, respectively. The mean number of parasites per fish did not significantly differ between fish held at the two temperatures and did not significantly decline over time at either temperature. Fish examined from 13 to 30 months postinfection all contained viable metacercariae that were morphologically and molecularly identified as B. damnificus. At 18 months, 12 metacercariae (of which 11 were intact and 10 displayed movement) were found in the one fish sampled; at 30 months, the last fish sampled contained three intact metacercariae (one displayed slight movement). Our results indicate that B. damnificus metacercariae can remain viable in channel catfish for at least an 18-30-month production cycle during which they have the potential to affect fish growth; in addition, infected fish may serve as intermediate hosts for these metacercariae for at least 2.5 years postinfection.

Use of Copper Sulfate to Control Egg Saprolegniasis at a Commercial Sunshine Bass Hatchery

AbstractAn obstacle to the larval production of sunshine bass (female White Bass Morone chrysops × male Striped Bass M. saxatilis) is fungal growth on the eggs caused by the water mold, Saprolegnia spp. Copper sulfate (CuSO4) is commonly used for fungus control in troughs at catfish hatcheries, but the effectiveness in other fish-egg hatching systems is unknown. Because sunshine bass eggs begin hatching after 48 h, CuSO4 treatments (0, 10, 20, and 40 mg/L) began the afternoon of spawning with a 10-min, aerated, static bath and were repeated the morning and afternoon of day 2; eggs were not treated after hatching began. An in vitro assay was developed for this treatment regimen that indicated maximum fungal inhibition was achieved with 20 mg/L CuSO4. In the effectiveness experiment in McDonald hatching jars (n = 3), fungus was severe in the untreated controls (27.7% survival), but very little fungus was present in treatments receiving 10 mg/L CuSO4 (31.4% survival) or higher. The control was not significan...

Comparative Effects of Copper Sulfate or Potassium Permanganate on Channel Catfish Concurrently Infected with Flavobacterium columnare and Ichthyobodo necator

A study was conducted to compare the effects of two chemical therapeutants on channel catfish (CCF) Ictalurus punctatus concurrently infected with Flavobacterium columnare and Ichthyobodo necator. Copper sulfate (CuSO4) and potassium permanganate (KMnO4) were investigated for their ability to reduce the bacterial load, parasite load, and subsequent mortality. Treatment rates of CuSO4 or KMnO4 were 2.1 mg/L and 3.0 mg/L, respectively, and were applied at 24 h intervals on three consecutive days and control fish were untreated. Fin and gill samples were taken on day 4 (24 h after the final treatment) and day 10 (one week after the final treatment) for quantification of parasite and bacterial load. The survival rate of CuSO4-treated fish (73.0 %) was significantly different from the untreated control fish (41.5%). KMnO4-treated fish was (53.6%) and not significantly different from untreated control or CuSO4-treated fish. I. necator loads were significantly reduced by both CuSO4 and KMnO4, but only CuSO4 significantly reduced the F. columnare load.

Comparison of Percent Hatch and Fungal Infestation in Channel Catfish Eggs after Copper Sulfate, Diquat Bromide, Formalin, and Hydrogen Peroxide Treatment

Abstract Reduced survival of fish eggs is often a result of infestation with fungi Saprolegnia spp. However, timely chemical treatments often limit these infestations and increase survival. The effect of copper sulfate pentahydrate (CSP; 10 mg of CSP/L of water), diquat bromide (25 mg of diquat cation/L), formalin (433 mg/L), and hydrogen peroxide (H2O2; 250 mg/L) on percent hatch and fungal infestation in eggs of channel catfish Ictalurus punctatus was compared in two identical trials (five replicates for each chemical treatment per trial). The percent hatch in all chemical treatments was significantly better than percent hatch of the controls. The amount of fungal coverage on egg masses treated with CSP, formalin, and H2O2 was significantly less than that observed on the controls. Although not statistically different from the other treatments, H2O2 at 250 mg/L exhibited the highest average percent hatch (64%; control hatch = 34%), the lowest fungal coverage (1.5 cm; control coverage = 7.2 cm), and the l...

Catfish Mucus Alters the Flavobacterium columnare Transcriptome

&NA; Columnaris disease, caused by Flavobacterium columnare, severely impacts the production of freshwater finfish species. Therefore, efforts to better understand the biological processes of F. columnare, including the formation of biofilms and their contribution to disease, are ongoing. In this study, we incubated F. columnare cultures with channel catfish mucus and used high‐throughput RNA sequencing to evaluate global changes in gene expression. Our data show that mucus activates in vitro biofilm formation. The analysis of F. columnare transcriptomes after the addition of mucus revealed significant differentially expressed genes (DEGs) between the planktonic and biofilm states. DEGs common among all biofilms were enriched for gene ontology groups including signal transduction, ligand binding and cellular homeostasis and are likely necessary for biofilm formation. Iron acquisition systems included TonB‐dependent receptor and ferroxidase genes were expressed among all biofilms, while siderophore synthesis genes were only expressed in mucus‐stimulated biofilms. The current analysis of F. columnare transcriptomes adds valuable information about the basic biological processes that occur during the planktonic and biofilm states. This work serves as a basis for future studies on understanding how biofilms are established and how they contribute to disease progression.