Development and comparison of loop-mediated isothermal amplification with quantitative PCR for the specific detection of Saprolegnia spp.

Saprolegniasis is an important disease in freshwater aquaculture, and is associated with oomycete pathogens in the genus Saprolegnia. Early detection of significant levels of Saprolegnia spp. pathogens would allow informed decisions for treatment which could significantly reduce losses. This study is the first to report the development of loop-mediated isothermal amplification (LAMP) for the detection of Saprolegnia spp. and compares it with quantitative PCR (qPCR). The developed protocols targeted the internal transcribed spacer (ITS) region of ribosomal DNA and the cytochrome C oxidase subunit 1 (CoxI) gene and was shown to be specific only to Saprolegnia genus. This LAMP method can detect as low as 10 fg of S. salmonis DNA while the qPCR method has a detection limit of 2 pg of S. salmonis DNA, indicating the superior sensitivity of LAMP compared to qPCR. When applied to detect the pathogen in water samples, both methods could detect the pathogen when only one zoospore of Saprolegnia was present. We propose LAMP as a quick (about 20-60 minutes) and sensitive molecular diagnostic tool for the detection of Saprolegnia spp. suitable for on-site applications.

Growth inhibition of Aeromonas salmonicida and Yersinia ruckeri by disinfectants containing peracetic acid.

Peracetic acid (PAA) is a therapeutic agent used for disinfection in aquaculture, but it must be investigated thoroughly in order to mitigate diseases without harming the fish. Successful disinfectants (like PAA) should not leave dangerous residues in the environment in order to successfully contribute to sustainable aquaculture. The aim of our study was to compare the effectiveness of 6 commercial PAA products with different molecular PAA:H2O2 ratios to reduce bacterial growth of Aeromonas salmonicida and Yersinia ruckeri and to determine effective concentrations and exposure times. All products reduced colony-forming units (CFUs) of A. salmonicida and Y. ruckeri. Products with higher molecular PAA:H2O2 ratios inhibited growth better than products with lower molecular PAA:H2O2 ratios at the same PAA concentration; this indicates that H2O2 is not the driving force in the reduction of A. salmonicida and Y. ruckeri growth by PAA in vitro. The practical application of the products with high molecular PAA:H2O2 ratios should be prioritized if these pathogens are diagnosed.

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.

Putative roles for a rhamnose binding lectin in Flavobacterium columnare pathogenesis in channel catfish Ictalurus punctatus.

Columnaris disease, caused by the bacterial pathogen Flavobacterium columnare, continues to be a major problem worldwide and commonly leads to tremendous losses of both wild and cultured freshwater fish, particularly in intensively farmed aquaculture species such as channel catfish. Despite its ecologic and economic impacts, the fundamental molecular mechanisms of the host immune response to this pathogen remain unclear. While F. columnare can induce marked pathologic changes in numerous ectopic tissues, the adhesion of F. columnare to the gill in particular is strongly associated with pathogen virulence and host susceptibility. Recently, in this regard, using RNA-seq expression profiling we found that a rhamnose-binding lectin (RBL) was dramatically upregulated in the gill of fish infected with F. columnare (as compared to naïve fish). Thus, in the present study we sought to further characterize and understand the RBL response in channel catfish (Ictalurus punctatus). We first identified two distinct catfish families with differential susceptibilities to columnaris disease; one family was found to be completely resistant while the other was susceptible (0% mortality versus 18.3% respectively, P < 0.001). Exclusively, in the susceptible family, we observed an acute and robust upregulation in catfish RBL that persisted for at least 24 h (P < 0.05). To elucidate whether RBL play a more direct role in columnaris pathogenesis, we exposed channel catfish to different doses of the putative RBL ligands l-rhamnose and d-galactose, and found that these sugars, protected channel catfish against columnaris disease, likely through competition with F. columnare binding of host RBL. Finally, we examined the role of nutritional status on RBL regulation and found that RBL expression was upregulated (>120-fold; P < 0.05) in fish fasted for 7 d (as compared to fish fed to satiation daily), yet expression levels returned to those of satiated fish within 4 h after re-feeding. Collectively, these findings highlight putative roles for RBL in the context of columnaris disease and reveal new aspects linking RBL regulation to feed availability.

The effect of high total ammonia concentration on the survival of channel catfish experimentally infected with Flavobacterium columnare.

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 E 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 x 10(5)) than did treatment 3 fish (1.2 x 10(7)), and treatment 2 fish (8.5 x 10(3)) had significantly less bacteria than did treatment 1 fish (6.9 x 10(4)), indicating that ammonia limited the F. columnare infection. The highest mean concentration of the bacteria (3.9 x 10(7)) 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.

Evaluation of continuous 4-day exposure to peracetic acid as a treatment for Ichthyophthirius multifiliis.

The parasitic ciliate Ichthyophthirius multifiliis infests all species of freshwater fish and can cause severe economic losses in fish breeding. The most effective treatment, malachite green, has been banned in Europe and North America for use in food fish production. Peracetic acid (PAA) was found to be toxic to I. multifiliis theronts at low concentrations. I. multifiliis-infested carp were exposed to 1 mg/l PAA in a dynamic exposure by means of peristaltic pumps. Five days after infestation, gills, tail fins, and skin below the dorsal fin were observed microscopically for I. multifiliis abundance. After PAA exposure, PAA-treated fish showed lower infestation of I. multifiliis in all investigated tissues than the unexposed control fish. The infestation increased in the control group whereas the infestation in the PAA-exposed groups significantly decreased (p = 0.0083, Bonferroni correction). The fish in the two exposure groups showed a slight reinfestation with I. multifiliis. This might be caused by a peroxide degradation (hydrolysis) and/or reduction of the delivered PAA concentration. Thus, PAA concentrations were possibly too low to be effective on the released trophonts and/or the infective theronts. This hypothesis is corroborated by the fact that the I. multifiliis in the gills, skin, and fins of the PAA-exposed carp were in an early developmental stage.

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.

Acute toxicity of peracetic acid (PAA) formulations to Ichthyophthirius multifiliis theronts.

Peracetic acid (PAA) is an antimicrobial disinfectant used in agriculture, food processing, and medical facilities. It has recently been suggested as a means to control infestations of Ichthyophthirius multifiliis. The purpose of this study was to determine the acute toxicity of two products containing 4.5% and 40% PAA to I. multifiliis theronts from two geographically separate isolates. Theronts were exposed to concentrations of PAA in 96-well plates containing groundwater at 23 degrees C. Acute toxicity was observed over a 4-h period. No significant difference in the median lethal concentration (LC(50)) estimates was evident between the two isolates at 4 h with the 4.5% PAA product (0.146 versus 0.108 mg/l PAA), while there was a statistical difference between the 4 h LC(50) with the 40% PAA product (0.274 versus 0.158 mg/l PAA). These results suggest that PAA is toxic to I. multifiliis theronts at low concentrations and that one of the isolates was more resistant to this compound.

Effects of Piperonyl Butoxide on the Metabolism of DEF S,S,S-Tributyl Phosphorotrithioate) in Fingerling Channel Catfish.

The present study was undertaken to investigate the significance of monooxygenases in bioactivation of DEF to a more effective anticholinesterase in fish. Channel catfish were exposed via the water column for 20 h to piperonyl butoxide (PBO) followed by a 4-h exposure to the organophosphate defolient DEF (concurrent with the PBO). Acetylcholinesterase (AChE) and aliesterases (ALiEs) activities were determined at 0 and 12 h after the exposure period. Inhibition of brain, liver, and plasma AChE activity by DEF was antagonized by PBO; muscle AChE was not inhibited by DEF. Piperonyl butoxide did not antagonize the inhibition of liver or plasma ALiEs by DEF. These results suggest that PBO retards the formation of the metabolite(s) of DEF that inhibit AChE, and that DEF is an effective inhibitor of ALiEs without metabolic activation.

Acute toxicity of isopropyl methylphosphonic acid, a breakdown product of sarin, to eggs and fry of golden shiner and channel catfish.

Several countries, including the United States, have agreed to destroy stockpiled chemical warfare agents in accordance with the Chemical Weapons Convention Treaty of 1993. Sarin is one of many chemical warfare agents (CWA) designated for destruction. In the event of an accident during incineration, sarin or its decomposition products have the potential to be expelled into the environment. Sarin hydrolyzes into isopropyl methylphosphonic acid (IMPA), a compound detected in groundwater from prior CWA production. This study determined the acute toxicity of IMPA to golden shiner, Notemigonus crysoleucas, and channel catfish, Ictalurus punctatus, eggs and 15-posthatch (dph) fry. The median lethal concentration (LC50) values at time of hatch for golden shiner and channel catfish eggs were 66.6 mg/L (hatched in 72 hr) and 167.5 mg/L (hatched in 168 h) IMPA, respectively. The 96-h LC50 estimates for 15-dph golden shiner and channel catfish fry were 93.9 and 144.1 mg/L IMPA, respectively. The lowest LC50 value from the most sensitive species in this study is approximately 100 times greater than the human adult lifetime drinking water health advisory value, and is approximately 2500 times greater than the critical reporting limit (> or =0.025 mg/L) for IMPA detection in groundwater from CWA production. These results are critical in understanding the toxicological properties of this potential environmental contaminant.

Hepatic microsomal desulfuration and dearylation of chlorpyrifos and parathion in fingerling channel catfish: lack of effect from Aroclor 1254.

Channel catfish were treated intraperitoneally with 100 mg Aroclor 1254/kg body weight and sacrificed at 96 h to observe the effects of this cytochrome P450 1A (CYP1A) inducer on chlorpyrifos and parathion metabolism. In the initial experiment, hepatic microsomal ethoxyresorufin-O-deethylase (EROD) activity of the Aroclor-treated fish was significantly induced but no effects on desulfuration or dearylation of chlorpyrifos or parathion were evident. In the second experiment, Aroclor 1254 did not alter total hepatic microsomal P450s content, but significantly induced hepatic EROD and CYP1A. There were no evident effects to other hepatic CYP isoforms recognized by anti-trout CYP2K1, CYP2M1 and CYP3A27. These experiments indicate that Aroclor 1254 did not induce the P450s responsible for metabolism of the phosphorothionate insecticides.