Involvement of cholinergic and adenosinergic systems on the branchial immune response of experimentally infected silver catfish with Streptococcus agalactiae

It has been recognized that the cholinergic and adenosinergic systems have an essential role in immune and inflammatory responses during bacterial fish pathogens, such as the enzymes acetylcholinesterase (AChE) and adenosine deaminase (ADA), which are responsible for catalysis of the anti‐inflammatory molecules acetylcholine (ACh) and adenosine (Ado) respectively. Thus, the aim of this study was to investigate the involvement of the cholinergic and adenosinergic systems on the immune response and inflammatory process in gills of experimentally infected Rhamdia quelen with Streptococcus agalactiae. Acetylcholinesterase activity decreased, while ACh levels increased in gills of infected animals compared to uninfected animals. On the other hand, a significant increase in ADA activity with a concomitant decrease in Ado levels was observed in infected animals compared to uninfected animals. Based on this evidence, we concluded that infection by S. agalactiae in silver catfish alters the cholinergic and adenosinergic systems, suggesting the involvement of AChE and ADA activities on immune and inflammatory responses, regulating the ACh and Ado levels. In summary, the downregulation of AChE activity exerts an anti‐inflammatory profile in an attempt to reduce or prevent the tissue damage, while the upregulation of ADA activity exerts a pro‐inflammatory profile, contributing to disease pathophysiology.     

Xanthine oxidase activity exerts pro‐oxidative and pro‐inflammatory effects in serum of silver catfish fed with a diet contaminated with aflatoxin B1

Several studies have associated the involvement of xanthine oxidase (XO) activity, a source of uric acid and reactive oxygen species (ROS), to pro‐oxidative and pro‐inflammatory effects during pathological conditions. Considering this, the aim of this study was to evaluate whether upregulation on seric XO activity may be a pathway involved in the oxidative stress in fish exposed to a diet contaminated with aflatoxin B₁ (AFB₁), as well as whether it may be considered a pathway involved in ROS and NOx production. Xanthine oxidase activity, as well as the uric acid, ROS and NOx levels increased in serum of fish fed with a AFB₁‐contaminated diet on days 14 and 21 post‐feeding compared to fish fed with a basal diet. Based on these evidences, upregulation of seric XO activity induces pro‐oxidant and pro‐inflammatory profiles in serum of fish fed with a AFB₁‐contaminated diet due to excessive formation on uric acid. Also, the excessive uric acid induces the release of pro‐oxidant and pro‐inflammatory mediators, as ROS and NOx, also contributing to oxidative and inflammatory profiles. In summary, the upregulation on seric XO activity may be considered a pathway involved in the oxidative stress of fish exposed to a diet contaminated with AFB₁.     

Involvement of xanthine oxidase inhibition with the antioxidant property of nanoencapsulated Melaleuca alternifolia essential oil in fish experimentally infected with Pseudomonas aeruginosa

Nanoencapsulated Melaleuca alternifolia essential oil (tea tree oil, TTO) is a natural alternative treatment, with 100% therapeutic efficacy in fish experimentally infected with Pseudomonas aeruginosa, and has also potent protective effects linked with antioxidant properties. However, the pathways responsible for the antioxidant capacity remain unknown. Thus, this study evaluated whether the inhibition of seric xanthine oxidase (XO) activity can be considered a pathway involved in the antioxidant capacity of nanoencapsulated TTO in fish experimentally infected with P. aeruginosa. Seric samples from fish infected with P. aeruginosa showed increased XO activity, as well as increased uric acid and reactive oxygen species (ROS) levels. In contrast, the prophylactic treatment with nanoencapsulated TTO prevented these infection‐induced alterations. Based on the evidence obtained, the upregulation of seric XO activity induced pro‐oxidative effects in the serum of fish experimentally infected with P. aeruginosa, due to excessive formation of uric acid, which stimulates the release of ROS. This treatment was able to prevent the upregulated seric XO activity and, consequently, the excessive formation of uric acid and ROS. In summary, inhibition of seric XO activity can be considered a pathway involved in the antioxidant capacity of nanoencapsulated TTO in fish experimentally infected with P. aeruginosa.     

Purinergic signalling displays an anti‐inflammatory profile in the spleen of fish experimentally infected with Aeromonas caviae: Modulation of the immune response

Extracellular adenosine triphosphate (ATP) and its metabolite adenosine (Ado) are recognized as key mediators of immune and inflammatory responses. Depending on its concentration, ATP may act as an immunostimulant or immunodepressant, while Ado levels display an anti‐inflammatory profile. The aim of this study was to evaluate whether splenic purinergic signalling is capable of modulating immune and inflammatory responses in fish experimentally infected with Aeromonas caviae. Triphosphate diphosphohydrolase (NTPDase) and 5′‐nucleotidase activities increased in the spleen of silver catfish (Rhamdia quelen) experimentally infected with A. caviae compared with the uninfected control group. Moreover, splenic Ado levels increased in the infected animals relative to the uninfected control group. Based on these lines of evidence, our findings revealed that adenine nucleotide hydrolysis is modified in the spleen of fish infected with A. caviae attempting to restrict the inflammatory process through the upregulation of NTPDase and 5′‐nucleotidase activities, which occurs in an attempt to hydrolyse the excessive ATP in the extracellular environment and rapidly hydrolyse AMP to form Ado. In summary, purinergic signalling can modulate immune and inflammatory responses during A. caviae infection.     

Blood–brain barrier breakdown and myeloperoxidase activity in silver catfish experimentally infected with Pseudomonas aeruginosa

Central nervous system (CNS) infections continue to be an important cause of morbidity and mortality, and microbial invasion of the blood–brain barrier (BBB) is considered a prerequisite for CNS infections, which contribute to behavioural abnormalities and disease pathogenesis. Based on this information, the aim of this study was to evaluate whether Pseudomonas aeruginosa causes disruption of the BBB, and to investigate the involvement of cerebral myeloperoxidase (MPO) activity in this process in experimentally infected silver catfish. The permeability of the BBB to Evans blue dye increased in the infected animals on days three and six post‐infection (PI) compared to the control group. Moreover, cerebral MPO activity and reactive oxygen species (ROS) levels also increased in the infected animals on days three and six PI compared to the control group. Based on this evidence, we concluded that P. aaeruginosa causes a disruption of the BBB, which may contribute to disease pathogenesis in the CNS. Moreover, the increase in cerebral MPO activity and ROS levels may be considered a pathway involved in BBB breakdown, allowing the passage of bacteria to the CNS.     

Efficacy of Different Salt (NaCl) Concentrations in the Treatment of Ichthyophthirius multifiliis-Infected Silver Catfish,Rhamdia quelen,Fingerlings

ABSTRACT

The objective of this study was to evaluate different salt concentrations in the treatment of Ichthyophthirius multifiliis. Silver catfish, Rhamdia quelen, fingerlings infected with I. multifiliis were submitted to four sodium chloride concentrations (g/L): 0,1,2, and 4 for 23 days. In a second experiment, fingerlings were maintained with 4 g/L salt, but for 45 days. Treatments with 2 and 4 g/L salt significantly increased fingerling survival compared with 0 and 1 g/L, and survival was significantly correlated with salt concentrations. In the second experiment, fish maintained at 4 g/L salt showed a gradual reduction of “white spots” and survival was 100%. Therefore, salt offers an alternative for treatment of silver catfish fingerlings infected with I. multifiliis.     

Citrus x aurantium essential oil as feed additive improved growth performance,survival, metabolic,and oxidative parameters of silver catfish (Rhamdia quelen)

The effects of dietary supplementation of graded level (0, 0.25, 0.5, 1.0, and 2.0 ml/kg diet) of Citrus aurantium essential oil (EOCA) on the growth, metabolic, and oxidative parameters of silver catfish (Rhamdia quelen) were investigated in a 60‐day growth trial. Fish fed with 2.0 ml EOCA per kg exhibited significantly better growth performance than those fed the control diet. Glucose, lactate, and protein levels in liver and muscle were altered significantly by dietary addition of EOCA. Hepatic lipid peroxidation levels, measured using thiobarbituric acid reactive substance and lipid hydroperoxides assays, were reduced in animals receiving the diet containing EOCA. Superoxide dismutase activity was higher, while glutathione S‐transferase activity was lower in the liver of fish receiving 0.5, 1.0, and 2.0 ml EOCA per kg of diet than in control. The nonprotein thiols content was higher in fish receiving the EOCA‐containing diet. Thus, dietary addition of EOCA improved growth, biochemical, and antioxidant parameters in silver catfish and could be useful as dietary supplement.     

Salt in the Food and Water as a Supportive Therapy for Ichthyophthirius multifiliis Infestation on Silver Catfish, Rhamdia quelen, Fingerlings

This study verified the efficacy of common salt (NaCl) in the food and water as a supportive therapy for Ichthyophthirius multifiliis and growth of silver catfish, Rhamdia quelen, fingerlings. After appearance of the first white spots following experimental exposure, indicating infestation by this parasite, fish were transferred to eighteen 40‐L polypropylene tanks and kept for 30 d. Experimental diets were prepared with five different levels of salt supplementation: 0.0, 1.2, 2.5, 5.0, and 6.0% for 30 d. In addition, another group of fish were fed a diet without salt supplementation but exposed to 4 g of salt/L of water. Fingerlings were collected at different times for analyses of whole‐body Na⁺, K⁺, and Cl^?. Dietary salt supplementation provoked a transitory increase in whole‐body Na⁺, K⁺, and Cl^? in infested catfish fingerlings, but the best stabilization of these ion levels was observed in specimens exposed to salt in the water. Silver catfish fed a diet containing 1.2% salt showed higher weight and biomass after 30 d compared to those fed with 6% salt. The use of 4 g of salt/L of water improved survival of infested silver catfish, but dietary salt supplementation was ineffective as a therapy for ichthyophthiriasis.     

Antiparasitic activity of the antimicrobial peptide HbβP‐1, a member of the β‐haemoglobin peptide family

A family of antimicrobial peptides (AMPs) derived from the β‐subunit of haemoglobin was recently isolated from channel catfish, Ictalurus punctatus, infected with Ichthyophthirius multifiliis (ich), an important freshwater fish parasite that causes ichthyophthiriosis. We previously discovered that one of these AMPs, HbβP‐1, had strong cidal activity against ich as well as another ectoparasite, Tetrahymena pyriformis. HbβP‐1 toxicity was specific, primarily affecting the trophozoite (trophont) stage of ich. Here, we show that HbβP‐1 acts more rapidly to kill smaller (presumably less mature) trophonts of ich, taking almost twice as long to kill larger trophonts (P < 0.0001). It acts more rapidly than an unrelated AMP, piscidin 1, which is haemolytic and also lethal to ich trophonts. HbβP‐1 is potently and selectively lethal to the trophont stage of the dinoflagellate ectoparasite, Amyloodinium ocellatum, one of the most important pathogens of warmwater marine fish. HbβP‐1 has no effect on the fish gill cell line feeder layer (G1B cells) used to propagate Amyloodinium, further suggesting a highly selective action. These findings suggest that HbβP‐1 or related AMPs might function in protecting marine as well as freshwater fish and that HbβP‐1 has highly selective activity against specific life stages of important fish ectoparasites.     

Aloysia triphylla essential oil as food additive for Rhamdia quelen – Stress and antioxidant parameters

We examined the effects of dietary supplementation for 21 days with different levels (0, 0.25 and 2.0 ml/kg) of the Aloysia triphylla essential oil (EOAT) on silver catfish (Rhamdia quelen). Cortisol levels were lower in fish fed EOAT at 2.0 ml/kg diet, and lactate levels were lower in those fed at both doses. Glucose, alanine aminotransferase, aspartate aminotransferase and triglyceride levels did not vary between groups. The biomarkers of oxidative stress, thiobarbituric acid‐reactive substances, lipid hydroperoxide, superoxide dismutase, catalase and non‐protein thiols, were lower in the brain, liver and muscle of fish fed EOAT at 2.0 ml EO/kg diet. Glutathione‐S‐transferase, reduced glutathione, plasma Na⁺, Cl^?, and K⁺, and gill Na⁺/K⁺‐ATPase and H⁺‐ATPase did not vary between groups. Taken together, our results clearly indicate that the addition of EOAT at 2.0 ml/kg diet improves oxidative status and lowers the stress response in silver catfish.     

Aeromonas caviae inhibits hepatic enzymes of the phosphotransfer network in experimentally infected silver catfish: Impairment on bioenergetics

Several studies have been demonstrated that phosphotransfer network, through the adenylate kinase (AK) and pyruvate kinase (PK) activities, allows for new perspectives leading to understanding of disease conditions associated with disturbances in energy metabolism, metabolic monitoring and signalling. In this sense, the aim of this study was to evaluate whether experimental infection by Aeromonas caviae alters hepatic AK and PK activities of silver catfish Rhamdia quelen. Hepatic AK and PK activities decreased in infected animals compared to uninfected animals, as well as the hepatic adenosine triphosphate (ATP) levels. Also, a severe hepatic damage was observed in the infected animals due to the presence of dilation and congestion of vessels, degeneration of hepatocytes and loss of liver parenchyma architecture and sinusoidal structure. Therefore, we have demonstrated, for the first time, that experimental infection by A. caviae inhibits key enzymes linked to the communication between sites of ATP generation and ATP utilization. Moreover, the absence of a reciprocal compensatory mechanism between these enzymes contributes directly to hepatic damage and for a severe energetic imbalance, which may contribute to disease pathophysiology.     

Effect of natural free gossypol and gossypol‐acetic acid on growth performance and resistance of channel catfish (Ictalurus punctatus) to Edwardsiella ictaluri challenge

A study was conducted to evaluate the effect of free gossypol from glanded‐cottonseed meal (G‐CSM) (natural free gossypol) or gossypol‐acetic acid on growth performance, body composition, haematology, immune response and resistance of channel catfish (Ictalurus punctatus) to Edwardsiella ictaluri challenge. Soya bean meal‐based diets supplemented with 0, 100, 200, 400, and 800 mg kg^?1 free gossypol from G‐CSM or gossypol‐acetic acid were fed to juvenile channel catfish in triplicate aquaria to apparent satiation twice daily for 12 weeks. Neither sources nor levels of dietary gossypol significantly influenced the final weight gain, feed intake, feed efficiency and survival of channel catfish. Similarly, whole‐body proximate composition, haematological parameters (red blood cell, white blood cell counts, haemoglobin and haematocrit), serum protein concentration, macrophage chemotaxis ratio, phagocytic activity and antibody production against E. ictaluri 21‐day postinfection were not significantly affected at either dietary sources or levels of gossypol. Gossypol concentrations of liver were linearly related to dietary level of gossypol but the retention rate varied dependent on sources of the dietary gossypol. At dietary gossypol levels of 400 or 800 mg kg^?1, total gossypol concentrations in liver of fish fed dietary gossypol from G‐CSM were significantly higher than those of fish fed the corresponding levels of gossypol from gossypol‐acetic acid. The (+)‐isomer of gossypol was predominantly retained in liver regardless of dietary sources of gossypol. The ratio of (+) to (?) gossypol isomers in liver decreased with increasing dietary concentrations of gossypol. Serum lysozyme activity of fish fed dietary gossypol levels of 200 mg kg^?1 or higher, either from G‐CSM or gossypol‐acetic acid, was significantly higher than that of the control. At a level of 800 mg kg^?1 diet, gossypol from G‐CSM stimulated significantly higher lysozyme activity than gossypol from gossypol‐acetic acid. Fish fed diets containing 400 mg kg^?1 gossypol or higher from G‐CSM or 800 mg kg^?1 gossypol from gossypol‐acetic acid had significantly increased superoxide anion (O) production. However, neither the sources nor the levels of dietary free gossypol influenced the resistance of juvenile channel catfish to E. ictaluri challenge.     

Effects of Water pH and Hardness on Infection of Silver Catfish,Rhamdia quelen,Fingerlings by Ichthyophthirius multifiliis

The aim of this study is to determine the effect of (1) different pH levels (5, 6, 7, 8, and 9) with water hardness 20 mg/L CaCO₃ and (2) pH 5 and 7 with varying water hardnesses (20, 60, and 120 mg/L CaCO₃) on the intensity of infection by Ichthyophthirius multifiliis in fingerlings of the silver catfish, Rhamdia quelen. In Experiment 1, the lowest and highest mortality rates were observed in fingerlings exposed to pH 5 and 9, respectively; in Experiment 2, fish kept at pH 5 and 7 with hardness 20 mg/L CaCO₃ showed significantly lowest and highest cumulative mortality, respectively, than those maintained in other conditions. The lowest intensity of trophonts/fingerling was observed at pH 5 and hardness 20 mg/L CaCO₃ in both experiments. We conclude that infection by I. multifiliis is less severe in silver catfish maintained at pH 5 and hardness 20 mg/L CaCO₃. High water hardness led to an increased intensity of trophonts and decreased survival in silver catfish kept at pH 5, but decreased intensity of trophonts and improved survival when the fish were kept at pH 7.     

Growth,immune response and resistance to Aeromonas hydrophila of darkbarbel catfish,Pelteobagrus vachelli (Richardson), fed diets with different linolenic acid levels

Experimental diets were formulated containing six levels of linolenic acid (0%, 0.5%, 1.0%, 1.5%, 2.0% and 2.5%) and each diet was fed to darkbarbel catfish Pelteobagrus vachelli in triplicate for 84 days. Weight gain, specific growth rate and feed efficiency increased with the increasing levels of dietary linolenic acid, but were not significantly different among diets containing 1.0%, 1.5%, 2.0% and 2.5% linolenic acid. Red blood cell count and haemoglobin were significantly higher in fish fed the 1.0% linolenic acid than other diets. Lysozyme activity and immunoglobulin M content in fish fed 1.0% of linolenic acid were significantly higher compared with the other diets. Antibody titre in fish fed 1.0–2.5% linolenic acids was significantly higher than in fish fed 0 or 0.5% linolenic acid 14 days after Aeromonas hydrophila challenge. This study indicates that linolenic acid is essential to darkbarbel catfish. Two‐straight broken‐line analysis with SGR showed that a dietary level of 1.29% linolenic acid is the minimum required for adequate growth of darkbarbel catfish. Considering other measured indicators of fish performance, a level of 1.0–1.5% linolenic acid in the diet is recommended for the darkbarbel catfish.     

Comparison of the lipid properties of healthy and pansteatitis‐affected African sharptooth catfish,Clarias gariepinus (Burchell), and the role of diet in pansteatitis outbreaks in the Olifants River in the Kruger National Park,South Africa

Pansteatitis has been identified in wild populations of sharptooth catfish, Clarias gariepinus (Burchell), and Nile crocodiles, Crocodylus niloticus Laurenti, inhabiting the same waters in the Olifants River Gorge in the Kruger National Park, South Africa. Mesenteric and pectoral fat tissue was investigated microscopically and by fatty acid analysis in healthy and pansteatitis‐affected catfish from both captive and wild populations. Variation in fatty acid composition between pectoral and mesenteric fat was noted. Composition of mesenteric fat differed between fish from various localities as a result of differences in diet. Pansteatitis in the captive population, resulting from ingestion of high amounts of dietary oxidized fat, reflected higher levels of unsaturated fatty acids within the mesenteric fat. Mesenteric fat of pansteatitis‐affected wild catfish was characterized by an increase in moisture content, a decrease in fat content and a decrease in stearic and linoleic acids. The n‐3 to n‐6 fatty acid ratio of mesenteric fat was higher in pansteatitis‐affected wild catfish than in healthy catfish from the same locality, reflecting higher polyunsaturated fat intake by pansteatitis‐affected fish. The possible role of alien, invasive, phytoplankton‐feeding silver carp, Hypophthalmichthys molitrix (Valenciennes), in the aetiology of pansteatitis in both catfish and crocodiles in the Olifants Gorge is discussed.     

Influence of native catfish mucus on Flavobacterium columnare growth and proteolytic activity

Flavobacterium columnare causes columnaris disease of farmed and wild freshwater fish. Skin mucus is an important factor in early stages of columnaris pathogenesis, albeit little studied. Our objectives were to (a) characterize the terminal glycosylation pattern (TGP) of catfish mucus, (b) determine the growth of F. columnare in formulated water (FW)‐containing channel catfish (Ictalurus punctatus) or hybrid catfish (Ictalurus punctatus X Ictalurus furcatus) mucus and (c) examine extracellular protease activity of two F. columnare isolates differing in virulence. The TGP of catfish mucus by lectin binding was as follows: alpha‐D‐mannose/alpha‐D‐glucose >N‐acetyl‐beta‐D‐glucosamine >N‐acetyl‐beta‐D‐glucosamine/N‐acetylneuraminic acid >N‐acetyl‐D‐galactosamine >alpha‐D‐galactose/N‐acetyl‐alpha‐D‐galactosamine >beta‐D‐galactose = alpha‐L‐fucose. Virulence studies demonstrated isolate AL‐02‐36 was highly virulent in channel catfish fry (0.1 g) with cumulative mortality of 90%‐100% versus 60% for isolate ALG‐00‐530 at equivalent doses (~3 × 10⁶ CFU/ml); a similar result was observed in larger (0.7 g) catfish. In multiple experiments, F. columnare replicated (2‐3 logs) and survived (28 days) in formulated water‐containing catfish mucus. Highly virulent isolate AL‐02‐36 possessed at least 2.5‐ to fivefold higher protease activity following growth in mucus than the less virulent ALG‐00‐530. Flavobacterium columnare utilized catfish mucus as a nutrient source and mucus presence modulated extracellular protease production.     

MH II‐DAB gene expression in grass carp Ctenopharyngodon idella (Valenciennes) after infection with the ciliate parasite,Ichthyophthirius multifiliis

The grass carp, Ctenopharyngodon idella (Valenciennes), is one of the most extensively aquacultured freshwater fish in China. However, because of the lack of effective control measures and the high‐density culture environment, considerable economic losses are caused by infection of C. idella with the parasitic ciliate, Ichthyophthirius multifiliis. The major histocompatibility (MH) DAB gene belongs to antigen‐presented genes in the class II genomic region, which is associated with parasite resistance. To understand the relationship of the DAB gene with I. multifiliis infection in grass carp, the expression profiles of MH II‐DAB were studied in tissues using real‐time quantitative polymerase chain reaction. The results showed that expression of the MH II‐DAB gene was up‐regulated in head kidney after I. multifiliis infection, and the expression peak appeared earlier in the study (case) group than in the control group. The obvious up‐regulation peak of MH II‐DAB gene was found at days 2 and 4 in skin; at 12 h to day 4 in spleen; at 12 h and days 1 and 6 in gill; and at day 10 in blood, whereas the MH II‐DAB gene was down‐regulated in liver and intestines after I. multifiliis infection. These results have implications for better understanding C. idella resistance to I. multifiliis infection.     

Effect of dietary iron (Fe) levels on growth performance,hepatic lipid metabolism and antioxidant responses in juvenile yellow catfish Pelteobagrus fulvidraco

This study was conducted to determine effects of dietary Fe levels on growth performance, hepatic lipid metabolism and antioxidant response for juvenile yellow catfish Pelteobagrus fulvidraco. Yellow catfish were fed six isonitrogenous and isolipidic diets containing Fe levels of 16.20, 34.80, 54.50, 76.44, 100.42 and 118.25 mg/kg for 8 weeks. Weight gain (WG) and specific growth rate (SGR) increased with dietary Fe levels from 16.20 to 54.50 mg/kg diet and then plateaued over the level. Feed conversion rate (FCR) was highest and protein efficiency rate (PER) was lowest for fish fed the lowest Fe levels of diet. Fe contents in whole body and liver increased with increasing dietary Fe levels. Hepatic lipid content was lowest, but mRNA levels of carnitine palmitoyltransferase (CPT‐1) and peroxisome proliferator‐activated receptor α (PPARα) were highest for fish fed 54.50 mg Fe/kg diet. Fish fed adequate dietary Fe levels reduced hepatic malondialdehyde (MDA) level and increased activities of antioxidant enzymes Superoxide dismutase (SOD), Catalase (CAT) and GS. Based on the broken‐line regression analysis of WG against dietary Fe levels, optimal dietary Fe requirement for yellow catfish was 55.73 mg Fe/kg diets. Fe‐induced changes in MDA levels and antioxidant enzymatic activities paralleled with the change in hepatic lipid content, suggesting the potential relationship between oxidative stress and hepatic lipid accumulation in yellow catfish.