Experiments on Virulence Dose and Portals of Entry for Aeromonas hydrophila in Walking Catfish

Abstract

Aeromonas hydrophila, isolated from chevron snakehead Ophicephalus (=Channa) striatus affected with epizootic ulcerative syndrome (EUS), was injected intramuscularly into healthy walking catfish Clarias batrachus at varying 10-fold serial dilutions from 10⁸ to 0 colony-forming units (cfu) per fish. Only 10⁶ or more cfu/mL induced dermomuscular lesions. Initial healing of lesions was observed by day 7 but complete healing was not apparent until day 16. Experiments were also conducted on possible portals of entry of A. hydrophila into walking catfish: Intramuscular (IM) injection, gastric gavage, fish food, and immersion of injured fish in rearing water inoculated with the test bacteria. Injuries were caused by skin or muscle cut, dermal scraping or incision, fish bite, and cohabitation of fish with golden snails Ampullarius sp. Only IM injection treatment induced dermomuscular pathology in the test catfish. This suggests that a localization of A. hydrophila to a level of 10⁶ cfu/mL in the musculature must be established for dermal lesions to develop.     

Electron Microscopy of Infection by Saprolegnia spp. in Channel Catfish

Abstract

Saprolegnia sp. isolated from channel catfish Ictalurus punctatus grew slower than S. parasitica on cornmeal agar (CMA). Oogonia in Saprolegnia sp. appeared frequently, whereas oogonia were rarely seen in S. parasitica on CMA. In experimental exposures of injured channel catfish to fungal spores, infections were apparent after 3–4 d, but were most common after 7–9 d. Multiple lesions were usually seen in naturally infected fish, whereas a single lesion appeared at the injured site of experimentally infected fish. No obvious differences were found between lesions caused by S. parasitica and those caused by Saprolegnia sp. Most of the epidermal cells in fungusinfected lesions were necrotic. In some lesions, the epidermis was completely sloughed and the dermis was exposed. Both Saprolegnia parasitica and Saprolegnia sp. penetrated the dermis, causing damage to fibroblasts and collagen lamellae.     

Effect of Temperature and Dissolved Oxygen Concentration on Edwardsiella ictaluri in Experimentally Infected Channel Catfish

Abstract

Channel catfish Ictalurus punctatus were experimentally infected with Edwardsiella ictaluri by immersion exposure. After clinical disease ran its course for 52 d, the surviving fish were exposed to one of the following environmental regimes in troughs: 25°C with aeration, 25°C with no aeration, or variable temperature (18–23°C) with no aeration. After 29 d of exposure to the environmental regimes, various organs and tissues of the fish were assayed to determine the effects of these conditions on E. ictaluri concentrations (colony-forming units/mL of tissue sample). The concentrations of this pathogen were significantly (P < 0.05) higher in all tissues (trunk kidney, liver, head kidney, blood, spleen, gallbladder, muscle, brain, and gonad) 52 d postinfection than 29 d after exposure to any of the environmental regimes (81 d postinfection). Fish exposed to a near-normal concentration of dissolved oxygen (6.4 mg/L) and a constant temperature of 25°C had E. ictaluri concentrations that were significantly (P < 0.01) lower than those offish exposed to a low oxygen concentration (2.6 or 1.8 mg/L) and either a constant or a variable temperature.     

The use of eugenol against Aeromonas hydrophila and its effect on hematological and immunological parameters in silver catfish (Rhamdia quelen)

The aim of this study was to evaluate the activity of eugenol against the fish pathogen Aeromonas hydrophila and eugenol's effect on hematological and natural immune parameters in silver catfish (Rhamdia quelen). In vitro, eugenol showed weak activity against A. hydrophila, but in vivo, at a subinhibitory concentration (10 mg L^?1), it promoted survival in infected silver catfish. Eugenol (50 μg mL^?1) reduced the hemolytic activity of A. hydrophila supernatant in vitro in fish erythrocytes. Subjecting catfish to eugenol baths (5 and 10 mg L^?1) for five days did not alter the hematological and immunological parameters studied in this work. Based on these results, eugenol can be used to treat or prevent bacterial diseases in fish.     

Aeromonas hydrophila Isolated from Wild Freshwater Fish in Croatia

Aeromonas hydrophila was recovered from fish living in lake Vrana on the Croatian island of Cres. The occurrence of the bacterium in the fish was assessed and related to gross signs of disease and findings at necropsy as a potential health hazard for fish. Isolated bacteria were subjected to morphological, physiological, biochemical and antibiotic susceptibility tests. A total of 26 A. hydrophila isolates were obtained. There was a clear seasonality, as no isolates were recovered in the summer months. Most of the isolates were sensitive to all the antimicrobials used in the study except novobiocin and penicillin G. Affected fish manifested haemorrhages over the skin, in the liver, kidney and swim bladder, spleen infarcts, fatty liver, ascitic fluid and swollen haemopoietic tissues. A. hydrophila does not appear to pose a major threat for the fish in the lake at present but under unfavourable and stressful conditions it could seriously compromise fish health.     

Effect of Dietary Lipid Sources on Production of Leukotriene B by Head Kidney of Channel Catfish Held at Different Water Temperatures

Abstract

Separate and combined effects of dietary fatty acids and water temperature on the production of leukotriene B (LTB) by the head kidney of channel catfish Ictalurus punctatus were investigated. Fish were fed semipurified diets containing 7% lipid as beef tallow, corn oil, linseed oil, menhaden oil, or a mixture (1:1:1) of menhaden oil, beef tallow, and corn oil. At 28°C, fish fed corn oil produced the greatest amount of LTB, and fish fed beef tallow produced the least. At 17°C, production of LTB by fish fed the beef tallow diet was again lowest; production of LTB by fish fed the corn oil diet was highest but was not significantly different from that obtained with the mixed-oil or menhaden oil diet. Production of LTB for the other diets was intermediate. This is the first report that the head kidney of channel catfish produces LTB; this production was affected by dietary lipid sources but not by the two water temperatures tested. Production of LTB was not related to weight gain. Moreover, the pattern of LTB production does not explain survival rates of channel catfish observed in a previous experiment in response to pathogen challenge. However, LTB concentration does appear to be roughly proportional to the amount of eicosanoid precursors (arachidonic acid and eicosapentaenoic acid) previously reported for liver phospholipids of channel catfish fed diets containing different amounts of essential fatty acids.     

Communications: Viability of Bacterial Pathogens in Frozen Fish

Abstract

Channel catfish Ictalurus punctatus injected with Aeromonas hydrophila, Pseudomonas fluorescens, Edwardsiella tarda, or E. ictraluri were frozen at ?20°C after death. Bacterial isolation at 2-d intervals after freezing indicated that A. hydrophila could be recovered for 20 d, P. fuorescens for 60 d, E. tarda for 50 d, and E. ictaluri for 30 d in frozen fish.     

Values for Selected Serum Analytes during Experimental Ichthyophthirius multifiliis Infection of Channel Catfish

Abstract

Channel catfish Ictalurus punctatus were exposed to a sublethal dose of infectious Ichthyophthirius multifiliis theronts, and eight serum analytes were measured. Sequential serum samples from each fish were collected for serum immediately before exposure, 4 d postexposure (PE), and 11 d PE. All fish developed moderately heavy infections with the parasite as indicated by the development of cutaneous lesions 5 d PE to infective theronts. Small but statistically significant changes in total protein, creatinine, and alkaline phosphatase values occurred after acute infection with I. multifiliis. Serum concentrations of glucose, albumin, urea nitrogen, alanine aminotransferase, and aspartate aminotransferase remained constant, indicating little disturbance of liver or kidney functions detectable by a serum biochemical profile established during an acute infection with I. multifiliis.     

Susceptibility of Selected Inland Salmonids to Experimentally Induced Infections with Myxobolus cerebralis,the Causative Agent of Whirling Disease

Abstract

Laboratory exposures to the infectious stages (triactinomyxons) of Myxobolus cerebralis demonstrated a range of susceptibility to whirling disease among four species of inland salmonids. Replicate groups of each species were exposed to two concentrations of triactinomyxons, a low dose (100–200 per fish) and a high dose (1,000–2,000 per fish). Exposed fish were evaluated for clinical signs, for severity of microscopic lesions at 35 d, 2 and 5 months, and for spore concentrations in the head cartilage at 5 months. A standard strain of rainbow trout Oncorhynchus mykiss matched for age served as a susceptible species control. Rainbow trout, westslope cutthroat trout O. clarki lewisi, Yellowstone cutthroat trout O. clarki bouvieri, and bull trout Salvelinus confluentus were susceptible to M. cerebralis infections. Clinical signs, including radical swimming (“whirling”) and black tails, were observed at 7 weeks postexposure among rainbow and cutthroat trout challenged at 3 weeks of age. Clinical signs were rare among bull trout exposed at an age of 4 weeks and absent among rainbow and cutthroat trout exposed at 3 months posthatch. Most rainbow, cutthroat, and bull trout were found to be infected when examined at 5 months postexposure. The most severe microscopic lesions among infected fish at 5 months postexposure were found among rainbow trout. Cutthroat trout had less severe lesions, bull trout had mild infections, and no evidence of infection was found among Arctic grayling Thymallus arcticus. Mean spore concentrations among infected fish correlated with the severity of microscopic lesion scores. Rainbow trout had mean concentrations of spores in head cartilage reaching 10⁶, whereas more resistant species such as bull trout had 10⁴ spores; no spores were found among Arctic grayling at 5 months postexposure.     

Subchronic Toxicity of Dietary Aflatoxin B1 to Channel Catfish

Abstract

Means for growth rate, hematocrit, hemoglobin concentration, and erythrocyte count of channel catfish Ictalurus punctatus fed 10,000 μg aflatoxin B₁ (AFB₁) per kilogram of feed for 10 weeks were significantly lower than those of fish fed 2,154 μg/kg or a lower concentration (P < 0.05). Mean leukocyte count was significantly higher in the fish fed the highest concentration of AFB₁ (P < 0.05). Gross appearance and behavior of all fish were normal. Histopathological effects were observed only in fish fed the highest concentration of AFB₁. These fish had foci of necrotic hepatocytes mixed with basophilic hepatocytes. Spaces, apparently resulting from hepatocellular necrosis, were present within the basophilic foci. Sinusoids in the head (hematopoietic) kidney were dilated and circular in profile. Increased hematopoietic activity of blood-forming tissues was apparent from the presence of numerous immature blood cells. The intestinal mucosal epithelium accumulated excessive amounts of iron pigments. Gastric glands in the stomach were necrotic and contained infiltrating macrophages.     

Longevity of Bolbophorus damnificus Infections in Channel Catfish

Abstract

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°C or 28°C. After 11 months, 6.8 ± 3.5 and 5.9 ± 3.0 metacercariae/fish were found in groups held at 18°C and 28°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.

Received July 14, 2010; accepted March 6, 2011     

Channel Catfish Response to Ultraviolet-B Radiation

Abstract

Fingerling channel catfish Ictalurus punctatus exposed to simulated ultraviolet-B radiation at an average daily dose of 2.9 J/cm² were quite sensitive to the radiation. After a 24-h exposure, thinning of the most dorsal epidermis frequently was accompanied by edema. Compared with epidermis of unexposed fish, mucous cells in exposed fish were less superficial and club cells were less numerous both dorsally and high on the lateral surface of the body. Sunburn cells with pyknotic nuclei were evident in the epidermis of exposed fish. Among fish exposed for 48 h, focal necrosis and sloughing of the outer epidermal layer were widespread. A methanol-extractable skin substance that is associated with resistance to sunburn in other fish species was not detected in channel catfish.     

Distribution of Edwardsiella ictaluri in California

Abstract

Wild and domestic populations of channel catfish Ictalurus punctatus were examined to determine the distribution of the disease called enteric septicemia of catfish (ESC) in California. The causative agent of ESC, Edwardsiella ictaluri, was isolated from five separate sites in California. Two of these isolations were from rectal swabs of asymptomatic fish, confirming that a carrier state may exist. Normal-appearing fish with serum antibody titer to E. ictaluri were commonly found in domestic channel catfish populations, suggesting that many fish become infected but recover. Wild channel catfish with antibody to E. ictaluri were also found in major reservoirs and water distribution canals. Edwardsiella ictaluri appears to be widely distributed within California.     

Communications: Experimental Production of Proliferative Gill Disease in Channel Catfish Exposed to a Myxozoan-Infected Oligochaete,Dero digitata

Abstract

Specific-pathogen-free fry of channel catfish Ictalurus punctatus raised in well water were exposed to Dero digitata (an oligochaete) collected from ponds where catfish have had proliferative gill disease (PGD) and where D. digitata is known to be infected with the triactinomyxid myxozoan Aurantiactinomyxon sp. These fry developed gill lesions and parasites characteristic of PGD. Fry exposed to suspensions of mature Aurantiactinomyxon spores obtained from squashes of infected D. digitata also developed PGD. Fry exposed to oligochaetes other than Dero spp., non-oligochaete benthic macroinvertebrates, or suspensions of squashes of D. digitata without identifiable myxozoans did not develop PGD.     

Responses of Blue Catfish and Channel Catfish to Environmental Nitrite

Abstract

In nitrite-exposure experiments, percent methemoglobin, plasma nitrite concentration, and plasma chloride ion concentration were compared between channel catfish Ictalurus punctatus and blue catfish I. furcatus exposed to sublethal levels of nitrite for 48 h at 25°C. In nitrite-recovery experiments, fish exposed to elevated environmental nitrite for 12 h were transferred to freshwater, and blood characteristics were monitored during the 24-h recovery period. Blue catfish appeared to be more resistant to environmental nitrite than channel catfish. Methemoglobin levels (percent of hemoglobin in methemoglobin form) were significantly lower in blue catfish than in channel catfish. Maximum plasma nitrite concentrations were 137 mg NO₂-/L plasma in blue catfish and 164 mg NO₂-/L plasma in channel catfish. Percent methemoglobin and plasma nitrite concentration were closely correlated. Plasma chloride decreased initially with exposure to nitrite but quickly returned to control levels. Blue catfish exposed to nitrite at 10°C required 1 week to recover when placed in nitrite-free water. The methemoglobin reductase enzyme apparently functioned at a slow rate in fish acclimated to cold temperatures.     

Blood Parameters and Immune Status of Rainbow Trout with Proliferative Kidney Disease

Abstract

Blood parameters, disease resistance, and the immune response were sequentially evaluated in rainbow trout Oncorhynchus mykiss with proliferative kidney disease (PKD). The fish were maintained under laboratory conditions, and the study group went through a full cycle of the disease. Hematological and serological changes occurred primarily in those fish with severe kidney lesions. Fish infected with the parasite that causes PKD demonstrated a greater resistance to bacterial challenge, and their immune responses were heightened when compared with those of uninfected fish. These data suggest that PKD alone is not a predisposing factor for secondary infections if the fish does not incur severe renal lesions.     

Communications: Specificity of the Channel Catfish Antibody to Edwardsiella ictaluri

Abstract

The specificity of channel catfish Ictalurus punctatus serum antibody to Edwardsiella ictaluri was characterized by microtiter agglutination assay. There was no correlation between antibody titer to Aeromonas hydrophila and antibody titer to E. ictaluri in wild or feral channel catfish. Anti-E. ictaluri antibodies in naturally infected channel catfish were not removed by adsorption by nine other species of bacteria found in the channel catfish intestine and fish ponds. Channel catfish immunized with nine other species of bacteria did not develop substantial antibody titer to E. ictaluri. The antibody response of channel catfish to E. ictaluri is highly specific, and the microtiter agglutination test is a specific indicator of previous exposure to E. ictaluri     

Chemotactic and Chemokinetic Responses of Channel Catfish Macrophages to Exoantigen from Edwardsiella ictaluri

Abstract

The ability of Edwardsiella ictaluri to attract macrophages of channel catfish Ictalurus punctatus was investigated. Exoantigen from E. ictaluri was tested for macrophage chemotactic activities both in vitro and in vivo. The exoantigen was chemotactic and chemokinetic for macrophages in vitro. Peritoneal injection of 750 μg of exoantigen protein into normal (E. ictaluri-free) channel catfish induced a marked increase in macrophage accumulations at 24 and 48 h. Neutrophil accumulation did not occur at the injection sites. Edwardsiella ictaluri exoantigen attracts macrophages, and this attraction may play an important role in macrophage responses during E. ictaluri infections.     

Effects of Dietary Zinc and Calcium on Select Immune Functions of Channel Catfish

Abstract

The effects of dietary zinc and calcium on immunocompetence of channel catfish Ictalurus punctatus (mean initial weight = 3.1 g/fish) were determined after a 10-week feeding trial. Fish were fed daily amounts equal to 3% of their body weight of one of six purified diets that were deficient in zinc (2 mg/kg of diet), calcium (0.02% of diet), or both; replete with zinc (20 mg/kg of diet) and calcium (0.5% of diet); or excessive in zinc (200 mg/kg of diet) or calcium (2.5% of diet). Immunocompetence of immunized and nonimmunized fish fed the various diets was assessed by measuring serum immunoglobulin M levels and mortality after an intraperitoneal injection of live Aeromonas hydrophila. Channel catfish fed diets deficient in zinc, calcium, or both had significantly (P ≤ 0.05) less weight gain and survival as compared with those fed diets replete with or excessive in zinc or calcium. Tissue mineral concentrations of fish indicated that various levels of zinc and calcium nutriture existed when immune functions were tested. Diet affected mortality of only nonimmunized fish in the bacterial-challenge test; zinc deficiency and calcium excess decreased mortality. Whereas growth and survival of fish were reduced by dietary zinc or calcium deficiency, the measured immunological characteristics were not equally impaired by these mineral deficiencies.     

Decreased Resistance to Edwardsiella ictaluri Infection in Channel Catfish Intraperitoneally Administered an Oil Adjuvant

Abstract

The effects of intraperitoneal injection of squalene, an oil adjuvant, on nonspecific mortality of channel catfish Ictalurus punctatus and on their resistance to experimental Edwardsiella ictaluri infection were studied. Yearling channel catfish were assigned to control (N = 22) or squalene (N = 25) treatment groups, and mortality was monitored for 14 d following treatment. On day 14 both groups were infected with E. ictaluri, the causative agent of enteric septicemia of catfish, and mortality was monitored for an additional 11 d. Before infection, mortality did not differ between groups. After E. ictaluri infection, fish that received squalene were at a substantially higher risk of dying than control fish (relative risk after squalene treatment = 6.86). These results suggest that intraperitoneal administration of squalene, although not directly toxic, decreased resistance to E. ictaluri infection.