Evaluation of diquat against an acute experimental infection of Flavobacterium columnare in channel catfish, Ictalurus punctatus (Rafinesque)

A trial was performed to evaluate the efficacy of diquat (6,7-dihydrodipyrido[1,2-a:2',1'-c]pyrazinediium dibromide) against an acute experimental infection of Flavobacterium columnare in channel catfish, Ictalurus punctatus . Diquat is an Environmental Protection Agency-approved herbicide and has the potential to be legally and practically used against columnaris. Channel catfish were challenged, by cutaneous abrasion, and waterborne exposure to F. columnare and treated once at 22-h post-challenge with 2.5, 5.0, 10.0 and 15 mg L⁻¹ of diquat active ingredient for 6 h. At the conclusion of the trial, 21-day post-challenge, diquat at 5.0, 10.0 and 15 mg L⁻¹ significantly ( P  < 0.05) reduced the mortality of infected fish from 95% in the challenged non-treated fish to 68%, 59% and 49%, respectively. In vitro , the minimum inhibitory concentration (MIC) of 23 isolates of F. columnare was assayed. The majority of the isolates had an MIC value of 5 μg mL⁻¹ (15 of the 23 isolates). Infected fish exhibited acute clinical signs similar to a natural infection. The skin had severe ulcerative necrotizing dermatitis and the muscles had severe necrotizing myositis. The gills had severe multifocal necrotizing branchitis. The results demonstrate that diquat would reduce mortalities caused by an acute columnaris infection.     

Acute columnaris infection in channel catfish, Ictalurus punctatus (Rafinesque): efficacy of practical treatments for warmwater aquaculture ponds

Columnaris disease was induced in channel catfish, Ictalurus punctatus (Rafinesque), by bath exposure to four highly virulent isolates of Flavobacterium columnare. In untreated controls, mortality began 20 h after exposure and reached 100% by 48 h. Mortality in channel catfish given antibiotic treatments with oxytetracycline or a combination of sulphadimethoxine and ormetoprim in feed prior to bacterial challenge was zero with all four strains of F. columnare. Diquat (Zeneca Agricultural Products, Wilmington, DE, USA) was the most effective bath treatment; mortality with all four strains was zero. With potassium permanganate, chloramine-T, hydrogen peroxide and copper sulphate, bath treatment efficacy varied significantly among strains (P = 0.0346) and among treatments (P = 0.0033). Bath treatments with chloramine-T and potassium permanganate significantly reduced (P < 0.05) mortality from 100 to 75 and 69%, respectively, but copper sulphate and hydrogen peroxide treatments were not effective. Based on our results, oral antibiotics prevented columnaris disease but, of the bath treatments, only Diquat produced a dramatic reduction in the mortality of acutely infected fish. Diquat is labelled for aquatic use as an herbicide in the USA but in large ponds it is prohibitively expensive.     

Larvae of Australian bass, macquaria novemaculeata (Steindachner 1866), can survive low salinities within 7 days of hatching

Abstract. Laboratory experiments were conducted on larvae or Australian bass, Macquaria novemaculeata (Steindachner), to establish the age at which they can fully osmoregulate in fresh water. The effect of acclimation at two different salinities (15‰ and 28‰) was also examined. It was found that larvae survived transfer to 2‰ salinity at 7 days post hatch, regardless of acclimation. Acclimation was also found to have little effect on the development of full osmoregulation in fresh water, which occurred between 21 and 28 days after hatching. The data obtained extend previous knowledge on the survival of larval Australian bass in low-salinity water and suggest that early release of suitably acclimated larvae into brackish ponds could reduce the need for intensive hatchery rearing.     

Influence of rearing conditions on Flavobacterium columnare infection of rainbow trout, Oncorhynchus mykiss (Walbaum)

The influence of rearing conditions on Flavobacterium columnare infection of rainbow trout, Oncorhynchus mykiss (Walbaum), was studied experimentally in the laboratory and at a fish farm. In experiment I, the effect of parasitic infection on columnaris disease was studied using F. columnare carrier fish. The fish were exposed to Diplostomum spathaceum cercariae and a set of other stressors in order to induce clinical columnaris infection. Parasitic infection and other stressors failed to induce the disease. Disease occurred when the fish were challenged with F. columnare, but D. spathaceum infection did not enhance the severity of the infection. In experiment II, the influence of rearing density and water temperature was studied. Overall mortality was highest in fish at normal rearing density with high temperature (+23 degrees C). At low temperature (+18 degrees C) mortality was not affected by rearing density, but the transmission of columnaris disease was faster at normal rearing density at both temperatures. This supports the view that reduction of fish density could be used in prevention of columnaris disease especially if water temperature is high. Because the lower rearing density can also decrease the transmission of ectoparasites and penetrating endoparasites, it could be an efficient tool in ecological disease management.     

Effects of varying salinities on the toxicity of potassium permanganate to larval and juvenile striped bass, Morone saxatilis (Walbaum)

Abstract. Eight-day-old striped bass, Morone saxatilus (Walbaum) larvae, and one-month-old juveniles were held in varying salinities and exposed to a range of concentrations of potassium permanganate. Lethal concentrations of potassium permanganate for 50% of the larval fish exposed for96h (96h LC₅₀) were 1middot;02, 2 middot;11, 1 middot;41 and 1 middot;73mg/1 for salinities of 0, 3, 6and 9 g/l, respectively; The 96h LC₅₀S for juveniles were 0 middot;96, 3 middot;26, 1 middot;63 and 1 middot;48 mg/l for salinities of 0, 5, 10 and 15g/l, respectively. There were significant differences in 96h LC₅₀ values at different salinities for both life stages. The 96 h LC₅₀ values derived for both life stages revealed a decrease in toxicity as salinity increased from 0 to 3–5g/l. Results indicated potassium permanganate to be toxic in fresh water to striped bass larvae and fingerlings at recommended treatment levels (3 middot;0mg/l).     

Kaolinitic clay protects against Flavobacterium columnare infection in channel catfish Ictalurus punctatus (Rafinesque)

Columnaris disease, caused by the bacterial pathogen Flavobacterium columnare, continues to be a major problem worldwide in both wild and cultured freshwater finfish. Despite the far-reaching negative impacts of columnaris disease, safe and efficacious preventatives and curatives for this disease remain limited. In this study, we evaluated the potential of kaolin (Al₂Si₂0₅(OH)₄), a type of clay, for the prevention of columnaris disease. Channel catfish, Ictalurus punctatus (Rafinesque), fingerlings were experimentally challenged with Flavobacterium columnare in untreated water or with water containing kaolin (1 g L⁻¹). Over the 7-day course of study, kaolin treatment led to significantly (P < 0.001) improved survival (96%) as compared to untreated fish (78% survival). Histological examination of the gills revealed that kaolin-treated fish had substantially less gill damage than untreated controls. Quantitative PCR analysis of gill tissue revealed that kaolin significantly reduced F. columnare adhesion (measured at 1 h post-challenge) and colonization (24 h post-challenge). Incubation of kaolin with F. columnare in vitro demonstrated that kaolin reduced the number of F. columnare cells in culture supernatants, presumably through the formation of physical complexes through adsorption. In summary, kaolin can improve survival, reduce gill pathologies and reduce bacterial attachment to key tissues associated with columnaris disease in channel catfish by binding to F. columnare.     

Salinity and temperature effects on whole-animal thyroid hormone levels in larval and juvenile striped bass,Morone saxatilis

Whole-animal thyroxine (T₄) and 3,5,3′-triiodothyronine (T₃) levels were measured in larval and juvenile striped bass, Morone saxatilis, reared for 10 days at one of three levels of salinity (equivalent to fresh water (FW), one-third seawater (1/3 SW), and seawater (SW) and two temperatures (15°C and 20°C). The striped bass were pre-metamorphic larvae, metamorphic larvae or juveniles. The short-term effects of seawater on plasma T₄ levels of juvenile striped bass were also measured. Higher salinities increased T₄ levels in premetamorphic larvae. In metamorphic larvae, SW and 1/3 SW increased T₄ levels and SW increased T₃ levels at 20°C. This response was eliminated in those at 15°C. Whole-animal thyroid hormone content was unaffected by salinity or temperature in juvenile striped bass, although significant fluctuations in plasma T₄ levels occurred in those transferred to 1/3 SW and SW. The thyroid axis of striped bass responds to salinity and temperature as early as in the pre-metamorphic stage. Thyroid hormones may mediate the beneficial effects of salinity on larval striped bass growth and survival.     

The influence of salmon surface mucus on the growth of Flavobacterium columnare

Flavobacterium columnare is the causative agent of columnaris disease. The presence of lesions on the gills, skin and fins of diseased fish suggests that F. columnare is able to utilize fish skin mucus as a substrate for growth and that exposure to this material would alter the expression of genes involved in the colonization of the outer surfaces of the fish. Growth, biofilm formation, extracellular protease production and changes in protein expression of F. columnare strain C#2 cultured in media supplemented with juvenile Atlantic salmon skin mucus were compared with the same media without mucus. C#2 was able to grow by using mucus as the sole nutrient source. Growth in mucus-containing media induced cells to grow as a biofilm and extracellular protease activity increased in mucus-containing cultures. SDS-PAGE protein profiles showed that expression of six extracellular proteins increased in mucus-containing media. These results demonstrate that salmon surface mucus promotes the growth of F. columnare and that exposure to mucus alters the growth characteristics of this bacterium with regard to protease production and biofilm formation. Further characterization of mucus-induced physiological changes will increase our understanding of the basis of virulence of this economically important fish pathogen.     

Food intake,growth, food conversion,and body composition of catfish exposed to different salinities

The effects of different salinities (0, 2, 4, 6 and 10‰) on food intake, growth, food conversion, and body composition of the freshwater catfish Mystus vittatus (Bloch) were studied. Under a restricted feeding schedule daily intake of food was found to be salinity dependent. Fish reared in 10‰ consumed more Tubifex tubifex, converted less efficiently and displayed poor growth as compared to individuals reared in fresh water. Fish flesh production decreased from 483 g (fresh water) to 177 g (10‰ salinity) as the salinity was increased. Water content of the fish was found to decrease with increase in salinity, while maximum ash (25.56%) and fat (42.25%) were exhibited by fish reared in 10‰ salinity.     

Morphological and genetic characteristics of Flavobacterium columnare isolates: correlations with virulence in fish

Variability in pathogenicity of Flavobacterium columnare makes disease treatment difficult because there is currently no way to easily recognize those strains that warrant aggressive treatments. In order to identify suitable virulence markers, 17 isolates of F. columnare were cultured from six different fish species. The DNA from all isolates was analysed using random amplified polymorphic DNA (RAPD). Bootstrap analysis of the RAPD data produced a tree with three major groups supported by bootstrap scores of 80-100%. Virulence of the isolates was determined by bath exposure of channel catfish, Ictaluruspunctatus (Rafinesque), and golden shiners, Notemigonus crysoleucas (Mitchill), to broth cultures of F. columnare. In channel catfish, 13 of 17 isolates produced 100% mortality within 48 h post-exposure. All isolates of cyprinid fish origin clustered in a single RAPD group. At least two of the four isolates that were not virulent in channel catfish were of cyprinid fish origin. There was a wide variation in cell morphology between isolates with lengths of cells or cell chains ranging from 3 to 11 microm, even under identical culture conditions. Most of the shorter or single cell isolates fell into a single RAPD group. No clear association was identified between virulence and any other characteristic, including RAPD group.     

柱状黄杆菌乙酰辅酶A合成酶基因及其上游调控序列分析

柱状黄杆菌(Flavobacterium columnare)是世界范围内危害淡水鱼类的柱形病的病原。目前对该病原菌遗传操作系统的研究进展较慢,而寻找高效稳定的启动子来调控外源基因在细菌体内的表达,有可能促进该细菌遗传操作系统的构建。本研究获得了柱状黄杆菌乙酰辅酶A合成酶基因(acetyl-coenzyme A synthetase gene,acs)的编码序列及其上游调控序列,该基因全长2 323 bp,编码635个氨基酸。通过序列分析,发现在该基因起始密码子ATG的上游存在核糖体结合位点(ribosome biding site,RBS)序列TAAAA,和启动子–7和–33的保守基序TATTTTCG和TTG。将acs的上游调控序列(promoter sequence,Pacs)置于氯霉素抗性基因(chloramphenicol acetyltransferase,cat)的上游并导入柱状黄杆菌G4株后,cat基因得以表达,并使宿主细胞产生稳定的氯霉素抗性。通过5′RACE技术,确定了外源的cat基因和内源的acs基因的转录起始位点都是位于起始密码子上游46 bp处的T。通过删减分析调控序列Pacs,发现起...     

Comparative challenge model of Flavobacterium columnare using abraded and unabraded channel catfish, Ictalurus punctatus (Rafinesque)

The early entry of the fish pathogen Flavobacterium columnare and enhancement by abrasion was studied in channel catfish, Ictalurus punctatus (Rafinesque), using the polymerase chain reaction and a species-specific primer set for a bacterial 16S rRNA gene product. Evaluations were conducted following an abrasion bath immersion challenge with F. columnare. Abrasion, a practice which has historically been used prior to bacterial challenge, had significant effects on the early entry of the pathogen and on cumulative percent survival (CPS). The FvpF1-FvpR1 primer set was useful in detecting the early entry of F. columnare in mucus, skin, gill, blood, liver and trunk kidney tissues in both abraded and unabraded fish following immersion challenge at 29 +/- 2 degrees C. Bacteria were detected earlier in all tissues in abraded fish, except in the trunk kidney. These differences were not significant, except in the case of blood. Mucus, skin and gill tissues were positive for F. columnare earliest regardless of treatment (after 5 min in abraded fish and after 15 min in unabraded fish). CPS following challenge with F. columnare was significantly affected by abrasion, which supports the use of abrasion for the F. columnare challenge model for channel catfish.     

Physiology of seawater acclimation in the striped bass,Morone saxatilis (Walbaum)

Several experiments were performed to investigate the physiology of seawater acclimation in the striped bass, Morone saxatilis. Transfer of fish from fresh water (FW) to seawater (SW; 31–32 ppt) induced only a minimal disturbance of osmotic homeostasis. Ambient salinity did not affect plasma thyroxine, but plasma cortisol remained elevated for 24h after SW transfer. Gill and opercular membrane chloride cell density and Na⁺,K⁺-ATPase activity were relatively high and unaffected by salinity. Average chloride cell size, however, was slightly increased (16%) in SW-acclimated fish. Gill succinate dehydrogenase activity was higher in SW-acclimated fish than in FW fish. Kidney Na⁺, K⁺-ATPase activity was slightly lower (16%) in SW fish than in FW fish. Posterior intestinal Na⁺,K⁺-ATPase activity and water transport capacity (J_v) did not change upon SW transfer, whereas middle intestinal Na⁺,K⁺-ATPase activity increased 35% after transfer and was correlated with an increase in J_v (110%). As salinity induced only minor changes in the osmoregulatory organs examined, it is proposed that the intrinsic euryhalinity of the striped bass may be related to a high degree of “preparedness” for hypoosmoregulation that is uncommon among teleosts studied to data.     

Hematology of great sturgeon (<Emphasis Type="Italic">Huso huso</Emphasis> Linnaeus, 1758) juvenile exposed to brackish water environment

The effect of environmental salinity on hematological parameters of great sturgeon Huso huso juveniles was studied. Five-month-old juveniles (mean body weight 28.3 ± 2.1 g) were subjected to 0, 3, 6, 9, and 12 ppt salinities. The hematological parameters were assessed after a period of 20 days rearing at these salinities. After transfer from fresh water to brackish water, red blood cells, hematocrit, haemoglobin and mean corpuscular haemoglobin decreased, but mean corpuscular volume increased. Mean corpuscular haemoglobin concentration, white blood cells, monocyte counts, and eosinophil counts showed no significant variations with increase in environmental salinity. An increase was found in lymphocyte counts according to the increase of salinity from 0 to 12 ppt, while the fresh water control group maintained basal levels. Decrease in neutrophil counts was observed in great sturgeon with increase in environmental salinity. These data show significant effect of salinity on the blood parameters of great sturgeon.     

Proteomic analysis of the sarcosine-insoluble outer membrane fraction of Flavobacterium columnare

Outer membrane proteins (OMPs) of bacteria are key molecules interacting with the host environment. Flavobacterium columnare, a pathogen-causing columnaris disease of fish worldwide, was studied in order to understand the composition of its OMPs. The sarcosine-insoluble membrane fraction of the OMPs was analysed using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) in combination with reverse-phase high-performance liquid chromatography-tandem mass spectrometry (RP-HPLC MS/MS). Thirty-six proteins were identified, including proteins involved in cell wall/membrane biogenesis, specific transport of various nutrients and in essential metabolism. The present study is the first report on the OMPs of F. columnare, and may serve as the basis for understanding the pathogenesis of the bacterium.     

Osmoregulatory changes in wedge sole (Dicologoglossa cuneata Moreau, 1881) after acclimation to different environmental salinities

The osmoregulatory responses of 20 days of acclimation to environmental salinities of 5‰, 15‰, 25‰, 35‰ and 55‰ were assessed in juveniles of wedge sole ( Dicologoglossa cuneata Moreau, 1881). This sole shows a good capacity to adapt to this range of environmental salinities. A direct linear relationship between environmental salinity and plasma osmolality was observed, with a calculated isosmotic point of 10.4‰ (284 mOsm kg⁻¹). Na⁺, K⁺-ATPase activity in the gills followed a 'U-shaped' relationship with environmental salinity, and a direct linear relationship in kidney tissue. Plasma cortisol levels were elevated in fish held in extreme salinities, and glucose levels were higher only in the group maintained at the highest environmental salinity. In the liver, a decrease in glycogen, lactate and amino acid contents was observed in specimens acclimated to extreme salinities (5‰ and 55‰), suggesting mobilization of liver metabolites. Metabolite levels in white muscle showed a pattern similar to the liver, with lower values in specimens acclimated to extreme salinities. We conclude that wedge sole is strongly euryhaline, but acclimation to extreme salinities comes with an energetic cost.     

A study on Flexibacter columnaris infection in loach, Misgurnus anguillicaudatus (Bleeker, Günther)

Abstract. Infectivity of Flexibator columnaris to loach, Misgurnus anguillicaudatus (Blecker, Günther), was studied with variation conditions and the presence of other bacteria in the water medium. Invasion of fish by the poathogen varied with different water conditions. The highest prevalence of infection was observed in an experimentally formulated water containing 0·03% NaCl 0·01% KCl, 0·002% CaCl₂2H₂O and 0·004% MgCI₂ 6H₂O. The percentage of infection in tap water was similar to that of the formulated water. The infeetion rate was low in the individual salt solutions, and no infection occurred in distilled water. Even in the formulated water, the pathogen failed to infect the fish when Citrobacter freundii, Aeromonas hydrophila or Pseudomonas fluorescens were present at densities 100 times that of F. columnaris. However, in such cases, the numbers of F. columnaris in the mucus of the fish body surface did not increase much throughout the experimental period. In contrast, F. columnaris suceessfully invaded the fish in the presence of either Streptococcus sp., Flavobacterium flavescens or Flavobacterium fuscum , even when the numbers of these bacteria were 1000 times that of F. columnaris. In this ease, F. columnaris gradually increased its numbers on the fish body surface.     

Experimental Infection of Rainbow Trout,Oncorhynchus mykiss,and Hybrid Striped Bass,Morone chrysops ♂ × Morone saxatilis ♀, with Viral Hemorrhagic Septicemia Virus Genotype IVb

The emergence of a new sublineage of viral hemorrhagic septicemia virus (VHSV) within the Laurentian Great Lakes has caused concern for aquaculture in the United States. Because of the occurrence of VHSV in a new geographic location, new host species have been identified and the complete host range has not been determined. This study confirmed the high resistance of rainbow trout, Oncorhynchus mykiss, to VHSV type IVb infection. In addition, the experimental susceptibility of hybrid striped bass, Morone chrysops ♂ × Morone saxatilis ♀, to VHSV type IVb infection was examined but determined to be highly dependent on age of fish and exposure temperature. No mortality was observed in adult fish infected via intraperitoneal (IP) injection at 15 C, whereas yearling fish infected via IP injection under the same conditions experienced 20.8% mortality. Among yearling fish infected via IP injection, mortality increased to 100% when exposure to VHSV occurred at 10 C. An LD₅₀ for yearling hybrid striped bass exposed to VHSV at 10 C by IP injection was determined to be 1.4 × 10⁴ pfu (SE = 2.1). Thus, at 10 C, yearling hybrid striped bass experience a high mortality when exposed to VHSV IVb by IP injection.     

Confinement and Transport-Induced Stress in White Bass Morone chrysops × Striped Bass M. saxatilis Hybrids: Effect of Calcium and Salinity1

Stress induced by severe confinement in cages and by simulated transport was evaluated in reciprocal white bass × striped bass hybrids (Morone chrysops × M. saratilis) after acclimation to various calcium concentrations in freshwater (5, 10, 20, 40, and 80 mg/L calcium) and various salinities (1, 8, 16, and 24%o) at 25 C. Survival was monitored in both confinement and transport events. In confinement events, changes in plasma osmolality were used as an indicator of osmoreg-ulatory dysfunction. After 6 h of severe confinement in cages, survival was significantly affected by treatments in both freshwater (FW) and saltwater (SW) events. Survival was greatest in 80 mg/L calcium in FW treatments and 8%o in the SW treatments. Simultaneous analysis of both FW and SW confinement data revealed that, after compensating for fish weight, survival increased as environmental ionic content increased (up to 8%o) then decreased with increasing salinity. Plasma osmolality decreased significantly from baseline levels with respect to confinement time in all FW treatments. Significant differences in plasma osmolality were observed among SW treatments: plasma osmolality of fish confined in 1%o decreased while those confined in salinities of 16 and 24%o increased. The plasma osmolality offish confined in 8%o remained near baseline levels. Almost no mortality (<5% in all treatments) occurred during (12 h) and after (5 d) transport in both FW and SW. Fish were in good condition and acclimated to transport water quality prior to transport events which may have been a major reason for high survival. Results from this study indicate that detrimental effects due to handling and transporting hybrid striped bass are reduced in FW environments that provide sufficient calcium and in SW environments which are near isoosmotic with the plasma.     

Effects of Nitrite Exposure on Growth and Survival of Sea Bass,Lates calcarifer,Fingerlings in Various Salinities

Sea bass, Lates calcarifer, fingerlings were acclimated to 0. 15, and 32 ppt, and the toxic effects of nitrite exposure were assessed. The 96-hour median lethal concentrations (96-hour LC₅₀ for nitrite were estimated to be 14.5 mg/L at 0 ppt, 105 mg/L at 15 ppt and 93 mg/L at 32 ppt salinity. Chronic exposure to a nitrite concentration equivalent to 10% of the respective 96 hour LC₅₀ resulted in marked growth reduction: growth being reduced in the order of 0 ppt > 32 ppt > 15 ppt. In nitrite-free water, growth rate for fish raised at a salinity of 15 ppt was higher compared to fish raised at salinities of 0 ppt and 32 ppt, a phenomenon which probably reflected the advantage of a reduction in osmoregulatory work in an iso-osmotic environment.