The immune response in rohu,Labeo rohita (Actinopterygii: Cyprinidae) to Argulus siamensis (Branchiura: Argulidae) infection: kinetics of immune gene expression and innate immune response

Argulus siamensis is a major pathogen in freshwater aquaculture. The immune responses of Indian major carp, Labeo rohita to experimental infection of A. siamensis was evaluated by quantitation of immune‐relevant gene expression in head kidney and skin, and serum innate immune parameters through the course of infection. In skin of infected fish, antioxidant genes like natural killer cell enhancing factor (NKEF‐B) and superoxide dismutase (MnSOD) were significantly up‐regulated in addition to lysozyme G and β₂ microglobulin (β₂M). Both tumour necrosis factor α (TNFα) and toll‐like receptor 22 (TLR22) genes were significantly down‐regulated in skin during early phases of the infection. Most of the genes exhibited significant down‐regulation in head kidney; immunoglobulin (IgM) and β₂M genes being the exceptions which were significantly up‐regulated at 12 h and 3 days post infection. Most of the innate immune parameters like serum complement activity and ceruloplasmin levels showed significant reduction in infected fish. The observed results are indicative of A. siamensis modulating the immune response of rohu by down‐regulation of many immune factors which may explain the susceptibility of rohu to A. siamensis infection. The interaction of this parasite with the host need to be further explored to understand its pathogenesis.     

Stress response of Salmo salar (Linnaeus 1758) facing low abundance infestation of Caligus rogercresseyi (Boxshall & Bravo 2000), an object in the tank,and handling

This study looks at how low infestation loads of adult Caligus rogercresseyi and other stressors affect the physiology of Salmo salar. Experimental fish groups were with (infested) or without (control) exposure to the parasite. The parasite cohort was followed for 78 days post‐infestation (dpi), and only adult lice were observed. Additional stressors were applied at 60 and 75 dpi. The analysis included measurements of fish physiology and weight. Low‐level infestations by adult C. rogercresseyi for more than 50 dpi induced moderate stress in S. salar as well as a high energy demand and increased small skin mucous cells. Threshold lice loads were identified, and above those loads, a high stress response was observed. Additional stressors altered fish physiology, inducing downregulation of the cortisol response after the first stressor and upregulation after the second stressor, but infested fish responded more strongly. Parasitism by C. rogercresseyi is energetically demanding, affecting the primary and secondary responses (e.g. cortisol and glucose levels), as well as the tertiary response (fish weight).     

Experimental infection of brown‐marbled grouper,Epinephelus fuscoguttatus (Forskal), with Vibrio parahaemolyticus identifies parvalbumin beta‐2 subunit I,alpha‐2‐macroglobulin,nattectin and immunoglobulin light chain,differentially expressed in resistant grouper

The mechanisms through which brown‐marbled grouper accomplishes resistance to infection, particularly against Vibrios, are not yet fully understood. In this study, brown‐marbled grouper fingerlings were experimentally infected with Vibrio parahaemolyticus, to identify disease resistance grouper, and the serum proteome profiles were compared between resistant and susceptible candidates, via two‐dimensional gel electrophoresis (2‐DE). The results showed that putative parvalbumin beta‐2 subunit I, alpha‐2‐macroglobulin, nattectin and immunoglobulin light chain proteins were among proteins that significantly overexpressed in the resistant fish as compared to the susceptible group of fish, whereas apolipoprotein E and immunoglobulin light chain proteins were observed to be differentially overexpressed in the susceptible fish. Further analysis by peptide sequencing revealed that the immunoglobulin light chain proteins identified in the resistant and susceptible groups differed in amino acid composition. Taken together, the results demonstrated for the first time that putative parvalbumin beta‐2 subunit I, alpha‐2‐macroglobulin, nattectin and immunoglobulin light chain are among important proteins participating to effect disease resistance mechanism in fish and were overexpressed to function collectively to resist V. parahaemolyticus infection. Most of these molecules are mediators of immune response.     

Evaluation of some intestinal cytokines genes expression and serum innate immune parameters in common carp (Cyprinus carpio) fed dietary loquat (Eriobotrya japonica) leaf extract

The aim of this study was to illustrate the effects of dietary loquat leaf extract (LLE) on the expression of some intestinal cytokines as well as serum innate immune parameters in common carp (Cyprinus carpio) fingerlings. The fish were fed with experimental diets supplemented with 0 (control), 0.25% (0.25LLE), 0.5% (0.5LLE) and 1% (1LLE) LLE over a 7‐week period. At the end of the trial, the serum lysozyme (Lys) activity, ACH50, total Ig as well as the expression of IL1B, IL8, TNF‐alpha, LYZ and TGF‐β in intestine were evaluated. The results showed that administration of 0.5% or 1% LLE significantly increased serum total Ig. However, in case of serum Lys activity significant elevation was observed just in fish fed 0.5% LLE. Also, supplementation of diet with LLE significantly increased ACH50 compared to the control group, regardless of inclusion levels. Gene expression studies revealed upregulation of TNF‐alpha, IL1B, IL8 and LYZ in intestine of fish fed LLE. However, the effects varied dependent on LLE levels and the tested immune related gene. Also, in case of TGF‐β significant downregulation was observed just in 1% LLE treatment. In conclusion, dietary LLE supplementation significantly upregulated immune related genes in intestine and improves innate immune responses. Altogether, LLE can be recommended as fish immunostimulant in early stage of carp culture.     

Effect of dietary supplementation of palm fruit extracts on the transcriptomes of growth,antioxidant enzyme and immune‐related genes in common carp (Cyprinus carpio) fingerlings

This study investigates the effects of date palm extracts [DPE] (Phoenix dactylifera L. Arecaceae) on growth, immune function and antioxidant system in common carp fingerlings (Cyprinus carpio). One hundred and twenty fish (4.06 ± 0.13 g) were divided into six groups fed on control diet or diets containing 200 mL kg^?1 DPE for 8 weeks. At the end of feeding trial, the expression of different genes was measured. Selected genes were grouped into three categories: growth factor genes in brain and liver [growth hormone (GH), insulin‐like growth factors I and II (IGF‐I and IGF‐II), antioxidant‐related genes in liver [glutathione S‐transferase‐alpha (GST‐α), glutathione reductase (GR) and glutathione peroxidase genes (GPX)] and immune‐related genes in head kidney [interleukin‐8 (IL‐8), interleukin‐10 (IL‐10) and transforming growth factor‐beta (TGF‐β) genes]. The relative expression of the growth‐related genes in fish fed DPE showed no significant increase compared to control group (P > 0.05). On the other hand, DPE altered the expression of genes encoding antioxidants enzymes in liver of fingerlings which was statistically significant with respect to the control samples in case GPX (P < 0.05). Also, DPE caused remarkable increases in the expression of the immune‐related genes (IL‐8, IL‐10 and TGF‐β) analysed on head kidney of common carp fingerlings compared to the control group (P < 0.05). In conclusion, it can be suggested that administration of DPE in early stages of common carp culture can promote immune efficacy and increase the antioxidant activity.     

Bromodeoxyuridine DNA labelling reveals host and parasite proliferation in a fish‐myxozoan model

Enteromyxum leei is a myxozoan parasite responsible for enteritis in gilthead sea bream (Sparus aurata). The parasite proliferates in the paracellular space of the intestinal epithelium and induces an inflammatory reaction. To assess intestinal cell turnover and parasite proliferation, fish were infected with the parasite by anal intubation; after 17 and 64 days, the cell proliferative marker bromodeoxyuridine (BrdU) was administered; and after 24 hr, tissue samples were taken for immunohistochemical detection. Parasite exposure induced increased epithelial and immune cell proliferation in all intestinal segments at all time points, even before parasite establishment. This increased turnover was triggered early after intubation and mainly at a local level, as shown by an increased proliferating cell nuclear antigen (pcna) gene expression only at the posterior intestine after 17 days (not found in lymphohaematopoietic organs). Incorporation of BrdU in parasite secondary and tertiary daughter cells indicated that parasite endogeny is not by schizogonial division, which uses de novo synthesis pathway of pyrimidines. Altogether, BrdU immunolabelling and pcna gene expression showed the rapid proliferative response of the fish intestines upon a myxozoan infection and how this response is effectively triggered even before the parasite reaches or establishes in the site.     

The experimental histopathology of Lernaea polymorpha Yu, 1938 infection in naive Aristichthys nobilis (Richardson) and a comparison with the lesion in naturally infected clinically resistant fish

Abstract. Bighead carp, Aristichthys nobilis (Richardson), fingerlings which had not been previously exposed to nauplii of Lernaea polymorpha were infected and sacrificed at various time intervals up to 21 days. Clinical observations showed that punctate haemorrhages developed following skin penetration at 13 days post-exposure. The adult female parasite was observed embedded in the skin and histopathology showed that the parasite penetrated the host tissue at an angle, sliding between the overlapping scales. It caused extensive tissue disruption, necrosis and haemorrhage along its path of entry, which was followed by an acute inflammatory response succeeded by a highly vascular chronic granulomatous fibrosis, whereby collagen fibres encapsulated the horns of the parasite. This contrasted with the predominantly haemorrhagic ulcerative lesions examined on immune fish subjected to natural infection, which were extensive but rarely accompanied by penetration through the dermis. There was a very extensive infiltrate of eosinophilic granular cells, club cells and lymphocytes within the spongiotic epidermis and the extensive haemorrhage was largely subepidermal.     

Alternate weekly exchanges of feeding regime affect the diversity of intestinal microbiota and immune status of Nile tilapia Oreochromis niloticus

The intestinal microbiota play an important role in the maintenance of the fish health. In this study, 150 Oreochromis niloticus were divided into five groups (G1–G5) and were subjected to alternate weekly exchange of feeding rate and frequency (3, 3, 1.5, 1.5 and 0%) and (2, 1, 2, 1 and 0 time), respectively, for 8 weeks. Enumeration of total intestinal bacteria revealed that the abundance of Lactobacillus was negatively correlated with those of Aeromonas, Pseudomonas and Edwardsiella. The abundance and proportion of Lactobacillus were affected by the change in feeding frequency. When fish were infected with Aeromonas hydrophila, survival rate significantly decreased in G5 (starved fish) compared with G1 (control group). The pro‐inflammatory cytokines, interleukin (IL)‐1β and tumour necrosis factor‐α, and anti‐inflammatory cytokine IL‐10 as well as serum antibacterial activity and respiratory burst activity were positively correlated with the proportion of Lactobacillus. The alternate weekly exchange of feeding regime did not alter the parametric morphology of the intestine, height and width of intestinal villi, number of goblet cells and width of muscle layer, except in G5. In conclusion, the alternate weekly exchange of feeding regime to starvation (G5), decrease in the feeding frequency to 1 time/day (G3) and decrease in feeding rate to 1.5% (G4) suppressed the immune status of fish, which became vulnerable to bacterial infection. Immune suppression was positively correlated with a decreased proportion of Lactobacillus spp.     

Molecular characterization,inductive expression and mechanism of interleukin‐10 gene induction in the Indian major carp,catla (Catla catla)

Interleukin‐10 (IL‐10) is a multifunctional cytokine and plays an important role in diseases. In this study, IL‐10 gene was cloned and characterized from catla (Catla catla), which is a highly commercially important fish species in the Indian subcontinent. The result indicated that the full‐length catla IL‐10 (cIL‐10) gene had five exons and four introns with an open reading frame of 540 nucleotides encoding a polypeptide of 179 amino acids. A phylogenetic analysis of cIL‐10 gene sequence showed that cIL‐10 clustered with freshwater carps group as expected. Quantitative real‐time polymerase chain reaction analysis showed that cIL‐10 was expressed in gill, liver, kidney, intestine, skin and heart and its expression profile was up‐regulated in bacterial infection and LPS treatment. A close relationship of high cIL‐10 expression and low pro‐inflammatory cytokine IL‐1β expression was observed in the treated group of fish, which might reveal the role of cIL‐10 as an anti‐inflammatory cytokine. Mechanism of cIL‐10 induction was investigated by blocking nuclear factor (NF)‐κB ‐signalling with BAY 11‐7082 in catla kidney cell culture. Blocking NF‐κB suppressed IL‐10 induction by LPS, and thus it revealed that cIL‐10 was induced through NF‐κB signalling. These data could be helpful to understand the function of IL‐10 in fish in response to vaccinations, probiotics and various diseases.     

Effect of guava Psidium guajava (L.) aqueous extract diet on growth performance,intestinal morphology,immune response and survival of Oreochromis niloticus challenged with Aeromonas hydrophila

An 84‐day feeding trial was carried out to evaluate the effects of aqueous Psidium guajava leaf extract (PGE) on growth, intestinal morphology, physiology, immune response and susceptibility of Oreochromis niloticus to Aeromonas hydrophila. Diets containing 0% (P0), 0.25% (P1), 0.50% (P2), 0.75% (P3) and 1.00% (P4) PGE were fed to triplicate groups of fish (mean weight; 1.32 ± 0.04 g) for 84 days. After the 84‐day feeding trial, test fish were injected with pathogenic A. hydrophila and then fed for 14 days. More feed were consumed in groups of fish fed PGE‐treated diets and resulted in significantly higher weight gain and feed intake. Incidentally, there was an increase in the calculated area of absorption of fish fed PGE diets, as accounted for by marginally higher villi length and width. Antioxidant and immune response were improved with PGE inclusion in diets as total protein, superoxide dismutase, glutathione peroxidase and glutathione S‐transferase significantly increased (p < 0.05) in fish fed PGE diets. Results of the challenge test with A. hydrophila revealed that the highest mortality (100%) was recorded in P0. This study revealed that inclusion of P. guajava extract in the diet of O. niloticus improved growth, nutrient utilization, immune system and survival of O. niloticus fingerlings.     

Quantitative investigation of Enteromyxum leei (Myxozoa: Myxosporea) infection and relative condition factor in cultured olive flounder Paralichthys olivaceus (Temminck and Schlegel)

Enteromyxum leei has been reported to cause emaciation disease in various fish species. To determine the effect of parasite intensity on cultured olive flounder Paralichthys olivaceus, we investigated the relationship between the relative condition factor (rCF = CF/standard CF × 100) and parasite load with quantitative polymerase chain reaction (qPCR) and the challenge test. A total of 57 cultured olive flounders were obtained from 11 fish farms and divided into five groups based on their rCF. We investigated the parasite intensity in the posterior intestine of the fish. The parasite load was closely matched to severe loss of body weight. In addition, olive flounders were inoculated either orally or anally with intestinal scrapings of infected fish or phosphate‐buffered saline. The fish were reared at natural water temperature and transferred to different tanks, and the water temperature was adjusted to 20°C after 6 weeks of inoculation. When the water temperature was increased to 20°C, the rCF decreased in the experimentally infected group. The results demonstrated that qPCR can be utilized to determine the relative abundance of E. leei in olive flounders and water temperature is an important factor to track the progress of the emaciation disease.     

Immunomodulation and interferon gamma gene expression in sutchi cat fish,Pangasianodon hypophthalmus: effect of dietary fucoidan rich seaweed extract (FRSE) on pre and post challenge period

A 45‐days feeding trial was conducted to study the immunomodulatory effect and interferon gamma gene expression of dietary fucoidan rich seaweed extract (FRSE) from Sargassum wightii on Pangasianodon hypophthalmus fingerlings. One hundred and eighty fingerlings were distributed into six experimental groups in triplicates. Each group was stocked with 10 fish and fed to satiation with iso‐nitrogenous (34.96 ± 0.09–35.18 ± 0.03 CP%) and iso‐caloric (368.65 ± 0.86–375.09 ± 0.26 Kcal/100 g) purified diets containing either 0% FRSE (control), 1% FRSE (TF₁), 2% FRSE (TF₂), 3% FRSE (TF₃), 3% seaweed powder (TS₃) or 6% seaweed powder (TS₆) in the feed. After feeding trial the experimental fish were challenged with Aeromonas hydrophila. Immunological parameters like respiratory burst activity, lysozyme activity, phagocytic activity and total leukocyte count (TLC) were increased with the increasing level of dietary FRSE, whereas serum Albumin/Globulin (A/G) ratio and blood glucose level exhibited decreasing trend (P < 0.05). Increased TLC, blood glucose level, respiratory burst activity, serum A/G ratio, lysozyme and phagocytic activities were recorded during the post‐challenge period. Maximum expression of interferon gamma (IFN‐γ) gene was recorded in FRSE fed groups than the control group both in pre and post challenge condition. After challenged with A. hydrophila the highest survival was observed in TF₂ and TF₃ groups whereas lowest survival was observed in the control group. Hence, dietary supplementation of FRSE at an optimum level of 2% reduced the stress and improved the immune status of P. hypophthalmus fingerlings.     

Effects of dietary soy isoflavones on growth,antioxidant status,immune response and resistance of juvenile grass carp (Ctenopharyngodon idella) to Aeromonas hydrophila challenge

The current study was conducted to evaluate the effect of dietary soy isoflavones (SI) on growth performance, antioxidant status, immune response and resistance to Aeromonas hydrophila in juvenile grass carp (Ctenopharyngodon idella). Six diets were formulated to contain 0 (control), 10, 50, 100, 500 or 1,000 mg SI per kg feed. Each diet was randomly allotted to triplicate net cages, and each net cage was stocked with 30 fish. The fish were fed one of the experimental diets to satiation twice per day for 60 days. The results showed that the WGR and DGC of the 500 mg/kg SI‐supplemented group were significantly higher than those of the non‐SI‐supplemented group (p < .05). Serum LZM and IgM activities in the SI‐supplemented groups were improved compared to the control group. SOD and GSH‐Px levels of fish fed the diet containing 500 mg/kg SI were significantly enhanced compared to those of fish fed the control diet (p < .05). Additionally, serum CAT, GSH‐Px and AKP activities in 50, 100 and 500 mg/kg SI‐supplemented groups were significantly higher than that in the control group (p < .05). The expression of most immune‐related genes (including IFN‐γ2, TNF‐α, M‐CSF2, IL‐6, IL‐12p40 and IL‐4) was significantly affected by dietary supplementation of SI. The group fed with 500 mg/kg SI had the highest 7‐day cumulative survival rate after challenge test (p < .05). The current results revealed that dietary inclusion of SI could improve the immune response and resistance against A. hydrophila and the supplementation level is suggested to be 500 mg/kg diet.     

Effects of dietary live or heat‐inactivated autochthonous Bacillus pumilus SE5 on growth performance,immune responses and immune gene expression in grouper Epinephelus coioides

To evaluate the possible dietary application of live and heat‐inactivated probiotic Bacillus pumilus SE5 in grouper Epinephelus coioides, juveniles (14.6 ± 0.2 g) were fed either a basal control diet (without probiotic) or the basal diet supplemented with 1.0 × 10⁸ CFU g^?1 live (T1) and heat‐inactivated B. pumilus SE5 (T2). The heat‐inactivated probiotic significantly improved the final weight, weight gain (WG) and specific growth rate (SGR) at day 60 and significantly decreased the feed conversion ratio (FCR) at day 30 and 60, while the viable probiotic significantly decreased the FCR at day 60 (P < 0.05). Phagocytic activity, serum complement C3 and IgM levels as well as SOD activity elevated significantly in fish fed the heat‐inactivated probiotic for 60 days (P < 0.05). Furthermore, the heat‐inactivated probiotic remarkably up‐regulated expression of TLR2 and pro‐inflammatory cytokines (IL‐8 and IL‐1β) in head kidney (P < 0.05), but the viable probiotic failed to do so. These results indicated that heat‐inactivated B. pumilus SE5 can effectively improve the growth performance and immune responses of E. coioides.     

Evaluation of the immune response in rainbow trout fry,Oncorhynchus mykiss (Walbaum), after waterborne exposure to Flavobacterium psychrophilum and/or hydrogen peroxide

The immune response in rainbow trout fry against Flavobacterium psychrophilum was elucidated using an immersion‐based challenge with or without prior exposure to hydrogen peroxide (H₂O₂). Samples were taken from the head kidney 4, 48, 125 and 192 h after immersion, and the regulation of several genes was examined. Bacterial load was assessed based on the presence of 16S rRNA and correlated with gene expression, and the levels of specific antibodies in the blood were measured 50 days post‐infection. Separately, both H₂O₂ and F. psychrophilum influenced gene expression, and pre‐treatment with H₂O₂ influenced the response to infection with F. psychrophilum. Pre‐treatment with H₂O₂ also affected correlation between gene regulation and pathogen load for several genes. A delay in antibody production in H₂O₂‐treated fish in the early phase of infection was indicated, but H₂O₂ exposure did not affect antibody levels 50 days post‐infection. An increasing amount of F. psychrophilum 16S rRNA was found in the head kidneys of infected fish pre‐treated with H₂O₂ relative to the F. psychrophilum group. The results show that a single pre‐treatment with H₂O₂ impairs the response against F. psychrophilum and may intensify infection.