Flavobacterium columnare genomovar influences mortality in channel catfish (Ictalurus punctatus)

Flavobacterium columnare, causal agent of columnaris disease, is pathogenic to many species of freshwater fish throughout the world. The United States channel catfish (Ictalurus punctatus) aquaculture industry is severely impacted by columnaris disease. The majority of the F. columnare isolates recovered from diseased channel catfish belonged to either genomovars I or II. The objective of the present study was to determine if differences existed in the ability of these genomovars to induce mortality in channel catfish. Single strand conformation polymorphism analysis (SSCP) was used to ascribe the isolates used in this study to the appropriate genomovar. Immersion challenge experiments (15min immersion exposure to approximately 5x10(5) to 1x10(6) CFU/mL) were carried out to assess virulence of genomovar I and II isolates to channel catfish. The results demonstrated that genomovar II (n=4) isolates were significantly (P<0.05) more virulent to channel catfish fry (92-100% mortality) than genomovar I (n=3) isolates (0-46% mortality). In vivo adhesion of the genetically characterized F. columnare also correlated (r2=0.73) to increased mortality in the challenged fry. In fingerling channel catfish, significantly higher mortality (P<0.05) resulted with genomovar II isolates ALM-05-182 and ALG-00-530 as compared to all the genomovar I isolates (n=3). Mortality of genomovar II isolate BGFS-27 with similar to genomovar II isolate (ALG-00-530) and two genomovar I isolates (ALM-05-53 and 140). The results suggest that although both genomovars are present in the aquatic environment, genomovar II appears to be more pathogenic for channel catfish.     

The in vitro effect of bovine lactoferrin on the activity of organ leukocytes in rainbow trout (Oncorhynchus mykiss), European eel (Anguilla anguilla) and wels catfish (Silurus glanis)

Lactoferrin (LF) is a glycoprotein found in milk, neutrophil granules, secretions and selected organs of mammals. Lactoferrin exhibits antibacterial, antiviral, fungicidal, immunoregulatory and other functions. Although fish are devoid of this protein and its cell receptors, LF effect on the immune mechanisms of fish has been demonstrated. The objective of this study was to investigate the effect of bovine lactoferrin, applied in vitro, on the activity of head kidney and spleen leukocytes in three freshwater fish species: rainbow trout (Oncorhynchus mykiss), European eel (Anguilla anguilla) and wels catfish (Silurus glanis). The obtained results validate LF beneficial effect on the respiratory burst of phagocytes in rainbow trout and wels catfish despite the fact that the potential killing activity against Aeromonas hydrophila was not stimulated in any of the studied species. Bovine lactoferrin enhanced the proliferation of T-lymphocytes in rainbow trout and European eel, as well as of B-lymphocytes in rainbow trout.     

In vitro evaluation of gut contractile response to histamine in rainbow trout (Oncorhynchus mykiss Walbaum, 1792)

The contractile response of intestinal strips in rainbow trout (Oncorhynchus mykiss Walbaum, 1792) to the administration of histamine was assessed by means of the organ bath technique. Intestinal strips were isolated from 16 clinically healthy fish and mounted in organ baths. Histamine was compared with the full agonist serotonin, to evaluate their contractile efficacy and potency. Serotonin elicited a concentration-related contraction in all examined intestinal strips, whereas histamine induced the contraction only in 14 exemplars. Of these, seven exhibited a concentration-related response. A sigmoidal curve was fitted from data (R(2) = 0.55) and its best fit values were compared with those of serotonin. Interestingly, histamine exhibited the same efficacy (E(max)) as serotonin (F-test, p > 0.05), but showed lower potency (by an order of magnitude) (F-test, p < 0.01). Moreover, the effect of the H1 antagonist, pyrilamine, has been tested to exclude aspecific contraction due to other agonists eventually released in situ following histamine administration. Pyrilamine showed a marked concentration-related antagonist action on the contractility induced by histamine with complete contractility antagonism at 10(-4)M. The authors suggest that the responses to histamine measured in the present study reflect a less sensitive response to an exogenous source of histamine, possibly due to bacterial metabolism.     

Plasma/muscle ratios of sulfadimethoxine residues in channel catfish (Ictalurus punctatus)

Channel catfish ( n = 84) maintained at a water temperature of 27°C were used in a feeding study to determine the plasma to muscle concentration ratios of sulfadimethoxine (SDM) and 4-N-acetylsulfadimethoxine residues. Sulfadimethoxine medicated feed was provided free choice at 42 mg SDM/kg body weight once daily for 5 days and the plasma and muscle concentrations of SDM were determined at selected withdrawal times (6, 12, 24, 48, 72, and 96 hours) following the last dose. Considerable variation in total SDM tissue concentration among fish within a sampling period was observed. For fish ( n = 12) at six hours post-dose, total SDM concentrations ranged from 1.4–24.8 μg/mL and 0.6–12.6 μg/g, with mean total SDM concentrations of 9.1 μg/mL and 5.3 μg/g for plasma and muscle, respectively. However, a mean plasma:muscle concentration ratio of 1.8:1 ± 0.3:1 was obtained over all concentrations and sampling periods. The plasma:muscle 95% t distribution interval for individual fish was 1.2:1 to 2.4:1. A correlation coefficient of 0.967 was obtained for the relationship between plasma and muscle total SDM concentration among individual fish ( n = 25). Results of this study indicate that plasma total SDM concentration may be used to identify samples containing violative SDM muscle residue. No fish contained total SDM muscle residues greater than the FDA tolerance (0.1 μg/g) by 48 hours following the final dose.     

Effect of vitamin E on carbonic anhydrase enzyme activity in rainbow trout (Oncorhynchus mykiss) erythrocytes in vitro and in vivo

Considering that the excessive usage of vitamin E causes hypervitaminosis and thus reduces blood erythrocyte concentrations, therefore it is worth studying how its pharmacological dosage affects the activity of carbonic anhydrase (CA) enzyme found in erythrocytes of rainbow trout (Oncorhynchus mykiss) in vitro and in vivo. Vitamin E inhibited CA enzyme and the IC50 value of the vitamin was 0.039 mM in vitro. Similarly, it was seen that vitamin E inhibited CA enzyme activity after the first hour following vitamin E injections in vivo. The activities of CA in groups of trout given vitamin E injection were measured at 1, 3 and 5 h and the corresponding activities were found to be 772.7 +/- 290.5 (P < 0.05), 1286.4 +/- 378.2 and 1005.7 +/- 436.1 enzyme units (EU) g Hb(-1). The difference over the control was significant (P < 0.05) in the first hour and insignificant at 3 and 5 h (P > 0.05). The activity of CA in the control, which did not contain vitamin E, was determined as 1597.7 +/- 429.0 EU g Hb(-1).     

Pathological effects of Arcobacter cryaerophilus infection in rainbow trout (Oncorhynchus mykiss Walbaum)

Arcobacter cryaerophilus was isolated from naturally infected rainbow trout (Oncorhynchus mykiss Walbaum), and its pathogenicity was tested by intramuscular injection into 40 healthy 1-year-old rainbow trout at 16 degrees C. The lethal dosage of 50% end point (LD50) for A. cryaerophilus was calculated 2.25 x 10(4) viable cells. Experimental infection caused deaths with gross clinical abnormalities such as degenerated opercula and gills, liver damage, haemorrhagic kidney and serous fluid in swollen intestines. The counts of A. cryaerophilus in kidney, liver and gills of experimentally infected fish ranged from 1.59 x 10(10) colony forming units (cfu)/g to 7.41 x 10(12) cfu/g. The means of erythrocyte (RBC) count, haematocrit level, serum cholesterol, triglyceride, albumin and total protein concentrations in the blood of the experimentally infected rainbow trout group were significantly lower than in the healthy fish. Leukocyte (WBC) counts of the experimentally infected rainbow trout were significantly higher than those of healthy fish. The present work shows that the selected blood characteristics may be good indicators of response to infections in rainbow trout.     

Influence of zearalenone on selected biochemical parameters in juvenile rainbow trout (Oncorhynchus mykiss)

Zearalenone (ZEA) is a mycoestrogen frequently found in food and animal feed materials all over the world. Despite its hydrophobic character, ZEA is also found in surface and ground waters which suggests an environmental risk for aquatic animals. Knowledge concerning mycotoxin-related mechanisms of toxicity is still incomplete, e.g. little is known about the influence of ZEA exposure on fish. The aim of this study was to investigate the effect of ZEA on selected biochemical parameters in juvenile rainbow trout after 24, 72, and 168 h of intraperitoneal exposure (10 mg/kg of body weight). The analysis showed a slight tendency towards prolonged blood clotting time and significant iron deficiency in the liver and ovary of exposed animals. However, no differences in aminotransferase (AlaAT, AspAT) activity or glucose levels in fish plasma was observed. The results of this study suggest that although trout exposed to ZEA did not exhibit any distinct symptoms of liver damage, the mycotoxin tested was able interfere with blood coagulation and iron-storage processes.     

Physiological and endocrinological responses during prolonged exercise in hatchery-reared rainbow trout (Oncorhynchus mykiss)

Rainbow trout (Oncorhynchus mykiss) were subjected to vigorous exercise (1.5 body length s-1), low exercise (0.5 body length s-1) or still-water (0.0 body length s-1). Hematocrit, glucose, growth hormone (GH), cortisol and triiodo-L-thyronine (T3) were monitored at the start of exercise, after 24 h, and after 2, 4, 8, 16 and 32 days of continuous swimming. Morphological indices and food intake were also monitored. At the end of the experiment, trout subjected to low exercise had gained significantly (p < 0.05) more weight than both the control (still-water) and vigorously exercised fish. This low exercised group of fish also consumed more food than the 2 other groups. Hematocrit increased significantly in both exercised groups at the onset of swimming but returned to pre-exercise levels within 8 hrs. Plasma glucose appeared to be generally unaffected by exercise. Likewise, plasma concentrations of both GH and T3 were not influenced by exercise. Plasma cortisol levels increased in an exercise dependent fashion at the onset of swimming, but returned to pre-swimming levels within 24 h and there was no apparent effect of sustained swimming. The results suggest: (i) the onset of exercise elicits transient changes in plasma components, (ii) the observed weight gain in low exercising salmonids occur without increases in circulating levels of GH or T3.     

Single intravenous and oral dose pharmacokinetics of florfenicol in the channel catfish (Ictalurus punctatus)

Plasma distribution and elimination of florfenicol in channel catfish were investigated after a single dose (10 mg/kg) of intravenous (i.v.) or oral administration in freshwater at a mean water temperature of 25.4 °C. Florfenicol concentrations in plasma were analyzed by means of liquid chromatography with MS/MS detection. After i.v. florfenicol injection, the terminal half-life (t(1/2)), volume of distribution at steady state (V(ss)), and central volume of distribution (V(c)) were 8.25 h, 0.9 and 0.381 L/kg, respectively. After oral administration of florfenicol, the terminal t(1/2), C(max), T(max), and oral bioavailability (F) were 9.11 h, 7.6 μg/mL, 9.2 h, and 1.09, respectively. There was a lag absorption time of 1.67 h in oral dosing. Results from these studies support that 10 mg florfenicol/kg body weight in channel catfish is an efficacious dosage following oral administration.     

In vitro evaluation of the susceptibility of Edwardsiella ictaluri, etiological agent of enteric septicemia in channel catfish, Ictalurus punctatus (Rafinesque), to florfenicol.

In vitro studies were conducted to assess the sensitivity of Edwardsiella ictaluri, the etiological agent of enteric septicemia of catfish (ESC), to the antibacterial drug florfenicol (FFC). Twelve different E. ictaluri isolates from cases submitted between 1994 and 1997 to the Thad Cochran National Warmwater Aquaculture Center fish diagnostic laboratory (Stoneville, MS) were used for testing. These isolates originated from channel catfish (Ictalurus punctatus) infected with E. ictaluri through natural outbreaks of ESC in the commercial catfish ponds in Mississippi. Seven hundred sixty-seven additional cultures of E. ictaluri were obtained from channel catfish infected experimentally with E. ictaluri. In some of these experimental infections, FFC was used for treatment. These cultures of E. ictaluri were identified by morphological and biochemical tests. Kirby-Bauer zones of inhibition (in mm) for FFC against E. ictaluri were determined using standard methods. The minimum inhibitory concentration (MIC) of FFC was determined for the natural outbreak E. ictaluri isolates and arbitrarily selected experimental cultures. The zones of inhibition for FFC tested with E. ictaluri ranged from 31 to 51 mm. The MIC for FFC tested with E. ictaluri was consistently 0.25 microg/ml. Edwardsiella ictaluri tested in these studies were highly sensitive to FFC in vitro.     

Localization of the initial developmental stages of Loma salmonae in rainbow trout (Oncorhynchus mykiss)

The intracellular microsporidian parasite Loma salmonae affects salmonids of the genus Oncorhynchus and is a significant cause of economic losses in pen-reared Chinook salmon (O. tshawytscha) in British Columbia. Loma salmonae infection is easily recognized by the xenomas that form in the gills, but early stages of infection are difficult to detect in histologic sections. In situ hybridization (ISH), using an L. salmonae-specific digoxigenin-labeled single-stranded DNA probe, was used to detect the parasite during the early stages of infection. Loma salmonae was detected in the gut mucosal epithelium as early as 24 hours postexposure (PE), and it localized in the lamina propria of the intestine within 24 hours of infection. After the parasite was detected in the lamina propria, dividing merogonic stages in infected cells in the heart were detected by ISH as early as 2 days PE, providing the first evidence of parasitaemia and hematogenous distribution of this parasite in infected blood cells. The parasites inside the infected cells appeared to be undergoing merogony as they passed through the heart, indicating that proliferation may start at the site of infection, before the parasite arrives to the gills for their final developmental phase. This is the first time that L. salmonae passage through the intestinal wall and migration to the heart has been visualized; however, the identity of the cells harboring the parasite has yet to be determined.     

Effect of alpha-tocopherol on antioxidant enzyme activities and lipid peroxidation in rainbow trout (Oncorhynchus mykiss)

In this study, the effect of alpha-tocopherol on the antioxidant capacity of rainbow trout (Oncorhynchus mykiss) was determined. For this purpose, activities of antioxidant enzymes such as catalase (CAT), peroxidase (POD) and glutathione reductase (GSSG-Rx) were investigated. Enzyme activities were measured at 0 (control), 1, 3 and 5 h after alpha-tocopherol injection. In addition, the levels of malondialdehyde (MDA) as a lipid peroxidation marker were determined in the erythrocytes. The results showed that alpha-tocopherol significantly activated the CAT, POD and GSSG-Rx enzymes as compared with the enzyme activities found in the controls (p < 0.05). However, MDA levels were significantly decreased by alpha-tocopherol treatment (p < 0.05). The results suggest that alpha-tocopherol may have a pro-oxidant tendency at a high dose and cause mild oxidative stress which could modulate signal transduction cascades, redirect gene expression, and influence many cellular responses such as proliferation, differentiation, and reproduction. For this reason, alpha-tocopherol should be used carefully in all applications in relation to fish.     

Acute handling disturbance modulates plasma insulin-like growth factor binding proteins in rainbow trout (Oncorhynchus mykiss)

The effects of acute stressor exposure on proximal (growth hormone [GH]) and distal (insulin-like growth factor-I [IFG-I] and insulin-like growth factor-binding proteins [IFGBPs]) components of the somatotropic axis are poorly understood in finfish. Rainbow trout (Oncorhynchus mykiss) were exposed to a 5-min handling disturbance to mimic an acute stressor episode, and levels of plasma GH, IGF-I, and IGFBPs at 0, 1, 4, and 24 h post-stressor exposure were measured. An unstressed group was also sampled at the same clock times (09:00, 10:00, 13:00, and 08:00 [the following day]) as acute stress sampling to determine temporal changes in the above somatotropic axis components. The acute stressor transiently elevated plasma cortisol and glucose levels at 1 and 4 h post-stressor exposure, whereas no changes were seen in the unstressed group. Plasma GH levels were not affected by handling stress or sampling time in the unstressed animals. Plasma IGF-I levels were significantly depressed at 1 and 4 h post-stressor exposure, but no discernible temporal pattern was seen in the unstressed animals. Using a western ligand blotting technique, we detected plasma IGFBPs of 21, 32, 42, and 50 kDa in size. The plasma levels of the lower-molecular-weight IGFBPs (21 and 32 kDa) were unaffected by handling stressor, nor were there any discernible temporal patterns in the unstressed animals. By contrast, the higher-molecular-weight IGFBPs (42 and 50 kDa) were affected by stress or time of sampling. Levels of the 42-kDa IGFBP levels significantly decreased over the sampling period in unstressed control animals, but this temporal drop was eliminated in stressed animals. Levels of the 50-kDa IGFBPs also decreased significantly over the sampling time in unstressed trout, whereas handling disturbance transiently increased levels of this IGFBP at 1 h but not at 4 and 24 h post-stressor exposure compared with the control group. Overall, our results suggest that acute stress adaptation involves modulation of plasma IGF-1 and high-molecular-mass IGFBP levels (42 and 50 kDa) in rainbow trout.     

Myxobolus cerebralis infection in rainbow trout (Oncorhynchus mykiss) and brown trout (Salmo trutta) exposed under natural stream conditions.

From early April into mid-June 1977, sequential groups of juvenile rainbow trout (Oncorhynchus mykiss) and brown trout (Salmo trutta) were each exposed for 10 days to the parasite Myxobolus cerebralis by immersion in a stream inhabited by infected wild trout. Following incubation in a M. cerebralis-free facility, trout were subsequently killed, and heads and gill arches were examined by routine histologic methods. A grading scale to quantify lesion severity was developed and applied. Percentage infected, lesion severity scores, effects of water temperature and flow rates on percentage infected and lesion severity scores, and resulting pathology were determined for each species at each exposure period. The percentage of rainbow trout infected with M. cerebralis was significantly higher than the percentage of brown trout infected for each exposure period. The percentages of rainbow trout infected in exposure periods later in the calendar year were significantly higher than those in earlier periods. The percentages of brown trout infected were not significantly different among exposure periods. Overall average lesion severity scores were significantly higher in rainbow than in brown trout. Lesion severity scores in rainbow trout increased over time (a positive correlation with exposure period). Lesion severity scores were not significantly different for brown trout among exposure periods. A significant correlation existed between water temperature and percentage of rainbow trout infected; a significant correlation also existed between water temperature and lesion severity scores in rainbow trout. Similar correlations did not exist for percentage of brown trout infected or accompanying lesion severity scores. In rainbow trout, ventral calvarium was the most common site of M. cerebralis replication, followed by gill arches. In brown trout, lesions were virtually confined to gill arches. Early lesions consisted of foci of cartilage necrosis with small numbers of M. cerebralis developmental stages. More advanced lesions consisted of multifocal areas of cartilage necrosis with numerous M. cerebralis developmental stages and/or mature myxospores bordered and/or infiltrated by mono- and multinuclear leukocytes. Lesions in brown trout were smaller and had fewer associated leukocytes and M. cerebralis developmental stages and/or mature myxospores. Higher infection rates, lesion severity scores, and differences in lesion location in rainbow versus brown trout explain in part why numbers of rainbow but not brown trout have fallen in western rivers inhabited with M. cerebralis-infected trout.     

Nonisotopic detection of Loma salmonae (microspora) in rainbow trout (Oncorhynchus mykiss) gills by in situ hybridization

Loma salmonae, a microsporidian parasite of salmonids of the genus Oncorhynchus, is a significant cause of economic loss in pen-reared chinook salmon (O. tschawytscha). Final stages of L. salmonae infections are easily recognized by the xenomas that form in the gills during sporogony. However, early prexenoma stages of infection (3 weeks or less after infection) are difficult to detect on histologic slides. An L. salmonae-specific single-stranded DNA probe labeled with digoxigenin was used to detect these prexenoma stages of L salmonae by in situ hybridization in experimentally infected rainbow trout. This method allows detection of the parasite in the gills only 2 weeks after infection, providing a sensitive and specific way of detecting L. salmonae during the early stages of infection.     

Proteomic analysis of rainbow trout (Oncorhynchus mykiss, Walbaum) serum after administration of probiotics in diets

The response of rainbow trout (Oncorhynchus mykiss, Walbaum) towards probiotics present in the feed was investigated by examining the proteome of serum as a measure of the acute phase response (APR). Proteomic analysis by two-dimensional electrophoresis (2D) concurrently with mass spectrometry was used to detect APR related proteins in rainbow trout serum following feeding with probiotics Aeromonas sobria GC2 and Bacillus sp. JB-1. Three candidate proteins increased following use of GC2, and were putatively identified as NADH dehydrogenase, dystrophin and mKIAA0350. Conversely, one of the proteins, which were induced following use of JB-1 was identified as transferrin.     

Effect of slaughter by removal from water on visual evoked activity in the brain and reflex movement of rainbow trout (Oncorhynchus mykiss)

The effects of removal from water and death by anoxia on brain function and reflex movement were examined in conscious trout. Visual evoked responses (VERS) were measured in fish with a body temperature of 20 degrees C, 14 degrees C and 2 degrees C. On average the fish took 2.6 minutes at 20 degrees C, 3.0 minutes at 14 degrees C and 9.6 minutes at 2 degrees C to lose VERS, and 11.5, 27.7 and 197.6 minutes, respectively, to lose all movement. VERS were absent from fish anaesthetised with MS222. From a humanitarian standpoint these results indicate that fish killed by anoxia could be exposed to periods of distress or suffering, and that cooling them in ice prolongs this period.     

Effects of long duration, low dose bronopol exposure on the control of Ichthyophthirius multifiliis (Ciliophora), parasitising rainbow trout (Oncorhynchus mykiss Walbaum)

Ichthyophthirius multifiliis Fouquet, 1876 infections on intensively reared fish stocks can increase rapidly, which if left unmanaged, can result in the heavy loss of stock. The present study explores the efficacy of long duration, low dose (1, 2 and 5 mg L(-1)) treatments of bronopol (marketed as Pyceze?, Novartis Ltd.) in reducing the number of trophonts establishing on juvenile Oncorhynchus mykiss held under small scale culture conditions. The effect of bronopol on the colonisation success of infective theronts was also investigated by adding 2 mg L(-1) bronopol to the water prior and during the infection process. The number of parasites surviving on fish treated this way was compared to groups of fish that only received treatment after infection had occurred. The effect of bronopol on exiting trophonts throughout their external development to the point of theront release was also assessed through the delivery of 1 mg L(-1), 2 mg L(-1) and 5 mg L(-1) bronopol for up to 27 days consecutively (days 9-36 post-infection). The trial showed that a nominal dose of 2 mg L(-1) bronopol administered prior to infection significantly reduced the number of theronts surviving in the water column at the time of the initial challenge by 35-40% (P<0.05). Similarly, doses of 2 and 5 mg L(-1) bronopol administered as the first wave of mature I. multifiliis trophonts exited fish (i.e. day 11 onwards) to develop externally, reduced the number of trophonts establishing on fish as the second cycle of infection by 52-83%. Continuous application of 2 and 5 mg L(-1) bronopol throughout the second and third cycles of I. multifiliis infection gave further reductions of between 90 and 98%. The number of trophonts on the fish in the control tanks and those treated with 1 mg L(-1) and the 2 mg L(-1) dose at the time of initial infection, by comparison, were observed to increase with successive cycles of infection. From these small scale tank trials, this study demonstrates that the strategic, long duration, low dose delivery of drugs like bronopol can significantly reduce the number of trophonts establishing on fish suggesting the potential of this drug at managing I. multifiliis infections.