David L. Straus

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.

Toxicity of peracetic acid (PAA) to tomonts of Ichthyophthirius multifiliis.

The free-living infective theront of Ichthyophthirius multifiliis historically has been thought to be the only stage susceptible to treatment. Here we introduce a technique to determine the toxicity of compounds to the newly released tomont, the encysted tomont and the developing tomites within the tomont that emerge as theronts. The toxicity of Wofasteril E400 (40% peracetic acid, PAA) to free-living forms of I. multifiliis was determined shortly after tomonts were physically removed from the surface of the fish and at 2.5 and 24 h after removal. Results indicate that 0.6 to 0.9 mg l(-1) PAA killed 39 to 82% of the newly released tomonts within 48 h when treated immediately. In a second experiment, tomonts were allowed to settle for 2.5 h after sampling from the skin and then treated for 12 h; concentrations > or =0.5 mg l(-1) PAA produced significantly fewer theronts than the controls. In a third experiment, encysted tomonts that were exposed to PAA 24 h after sampling from the skin and treated for 2 or 4 h produced a variable amount of theronts, but the concentrations tested (0.5 to 3.0 mg l(-1)) did not halt theront production. This research demonstrates that encysted I. multifiliis are less susceptible to chemical treatments.

Efficacy of Potassium Permanganate in Treating Ichthyophthiriasis in Channel Catfish

Abstract Epizootics of ichthyophthiriasis can be controlled with potassium permanganate (KMnO4), but its effectiveness has not been confirmed by controlled studies. The purpose of this study was to determine the concentration of KMnO4 needed to halt an active Ichthyophthirius multifiliis infestation in channel catfish Ictalurus punctatus. Juvenile channel catfish were exposed to fish infested with I. multifiliis until they developed immature trophonts. They were then moved to individual static containers with 2 L of filtered well water, where they were treated with KMnO4 daily for 10 d. The lowest effective dose of KMnO4 required to eliminate theronts was 1.25 mg/L. The results indicate that KMnO4 is effective for controlling I. multifiliis epizootics at low concentrations in clean water. However, effective treatment in ponds will be strongly influenced by detoxication of KMnO4 depending on the concentration of easily oxidizable substances in the water.

Prevention of an Initial Infestation of Ichthyophthirius multifiliis in Channel Catfish and Blue Tilapia by Potassium Permanganate Treatment

Abstract Potassium permanganate (KMnO4) has been used to control infestations of Ichthyophthirius multifiliis, but its effectiveness has not been reported from controlled efficacy studies. The purpose of this study was to determine the acute toxicity of KMnO4 to the I. multifiliis theront and the concentration needed to prevent an initial infestation of I. multifiliis in juvenile channel catfish Ictalurus punctatus and blue tilapia Tilapia aurea. Ichthyophthirius multifiliis theronts were exposed to concentrations of KMnO4 in 100 μL of well water in 96-well plates and observed for 4 h to determine the acute toxicity. A concentration of 0.9 mg KMnO4/L caused greater than 95% mortality of the theronts in 4 h in well water; the 4-h LC50 (concentration lethal to 50% of test animals) value was estimated to be 0.77 mg/L. Juvenile channel catfish were exposed to 10,000 theronts/L of well water and immediately treated with a single dose of KMnO4. Infestation occurred in controls 6 d after exposure. The lowest eff...

The acute toxicity of copper to blue tilapia in dilutions of settled pond water

Blue tilapia fingerlings (Oreochromis aureus) were exposed to copper (Cu) in a series of static toxicity tests. The water used in this study was composed of settled pond water or settled pond water diluted with deionized water. Estimates of mean 96-h LC50 values (median lethal concentration) were 43.06, 6.61, 0.69 and 0.18 mg/l Cu as copper sulfate) in waters having total alkalinities of 225, 112, 57 and 16 mg/l CaCO3, respectively. These data demonstrate the typical acute toxicity response of Cu, in which toxicity to tilapia increases as pH, total alkalinity and total hardness decrease. The results indicate that blue tilapia are relatively tolerant to Cu when compared to other species and that copper sulfate treatments in low-alkalinity waters may be detrimental to the health of blue tilapia.

Histological and Hematological Evaluation of Potassium Permanganate Exposure in Channel Catfish

Abstract A histological and hematological study was performed to evaluate the effect of waterborne exposures of channel catfish Ictalurus punctatus to potassium permanganate (KMnO4). Three concentrations of KMnO4 were chosen to represent one, three, and five times the therapeutic concentrations (0.438, 1.315, and 2.190 mg/L, respectively), based on the KMnO4 demand, for 36 h, which is three times the usual treatment duration. The organs examined were the gill, liver, and trunk kidney. Differential leukocyte counts of neutrophils and monocytes in the blood and plasma enzyme analyses (lactate dehydrogenase and alanine transaminase) were also performed. The gill was the only organ to show microscopic lesions. Fish exposed to the therapeutic concentration of KMnO4 for 36 h had mild hypertrophy and spongiosis in the gills sampled during exposure, but no lesions were noticed 2 d postexposure. Gills of fish exposed to three and five times the therapeutic dose had extensive hyperplasia, epithelial hypertrophy and...

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 potassium permanganate against an experimental subacute infection of Flavobacterium columnare in channel catfish, Ictalurus punctatus (Rafinesque)

An experiment was performed to evaluate the efficacy of potassium permanganate (KMnO4) as a prophylactic and therapeutic treatment of an experimental subacute infection of Flavobacterium columnare in channel catfish, Ictalurus punctatus. Fish were cutaneously abraded and divided into five treatment groups: (i) challenged by waterborne exposure to F. columnare and not treated with KMnO4 (positive control), (ii) challenged and simultaneously treated with KMnO4, (iii) challenged and treated with KMnO4 at 1, 6 and 9 days post-challenge, (iv) not challenged and treated with KMnO4 at 1, 6 and 9 days post-challenge (first negative control) and (v) not challenged and not treated (second negative control). The dosing of KMnO4 was 2.0 mg L(-1) above the potassium permanganate demand for 2 h duration. The survival of the group challenged and simultaneously treated with KMnO4 (99%) was significantly higher than the positive control (78%) and was not significantly different from the negative control groups. The challenged fish treated with KMnO4 post-challenge had 7% higher survival than the positive control (85% compared with 78%), but that difference was not statistically significant. The results demonstrate that KMnO4 has a clear prophylactic value but probably a marginal therapeutic value once the infection has established.

Optimizing copper sulfate treatments for fungus control on channel catfish eggs.

This range-finding study determined the optimum concentration of copper sulfate (CuSO4) for fungus control on eggs of channel catfish Ictalurus punctatus. The study consisted of five CuSO4 concentrations (2.5, 5, 10, 20, and 40 mg/L) and an untreated control in a flow-through system. A single spawn was used for each replication (N=4). Eggs were treated daily until the embryos reached the eyed stage. 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; survival of hatched fry in the control group was approximately 2%. The optimum CuSO4 treatment, as determined by percent survival of hatched fry, was 10 mg/L daily (69% survival); survival for this treatment group was significantly different from that for the controls. Very little fungus was present in treatments receiving 10-mg/L CuSO4 or higher except in one replication that had approximately 40% unfertilized eggs. The average survival rates in the 0-, 2.5-, 5-, 10-, 20-, and 40-mg/L CuSO4 treatments were 2, 34, 50, 69, 59, and 51%, respectively.

Copper sulfate toxicity to two isolates of Ichthyophthirius multifiliis relative to alkalinity

Theronts from 2 different strains of Ichthyophthirius multifiliis (AR1 and AR5) were exposed to copper sulfate (CuSO4) in waters of different total alkalinities and observed for 4 h to determine relative toxicity and kinetics of parasite mortality. Consistent with the known solubility properties of the metal, Cu was significantly more toxic to cells maintained under low (48 mg l(-1)) compared with high (243 mg l(-1)) total alkaliniity conditions. This was reflected in both the median lethal concentration (LC50) values and rates of mortality for both parasite strains; strain differences were also observed. The AR1 strain was significantly more resistant to copper toxicity than the AR5 strain in both high and low alkalinity waters. In general, these strain differences were more evident under conditions of low stress (i.e. low CuSO4 concentration and high alkalinity), and suggest that genetic factors are overridden under high stress conditions. The present study establishes a role for alkalinity in the effectiveness of CuSO4 treatment of ichthyophthiriasis and reveals differences in the susceptibility of parasite populations that are clearly important for control programs.