Heat-killed Aeromonas Hydrophila Research Articles

Innate Immune Response Assessment in Cyprinus carpio L. upon Experimental Administration with Artemia salina Bio-Encapsulated Aeromonas hydrophila Bacterin.

The present study aimed to analyze the enhancement of innate immune responses in juvenile-stage common carp (Cyprinus carpio L.), upon the administration of heat-killed Aeromonas hydrophila at a dosage of 1 × 107 CFU ml-1 through bio-encapsulation in the aquatic crustacean, Artemia salina. This work emphasizes the modulation of innate immune response when administered with the bio-encapsulated heat-killed antigen that acts as an inactivated vaccine against Motile Aeromonas Septicemia disease. Bio-encapsulated oral administration of antigens promotes innate immunity in juvenile-stage fishes. The optimization of effective bio-encapsulation of bacterin in Artemia salina nauplii was carried out and the best optimal conditions were chosen for immunization. The functional immune parameters such as myeloperoxidase, lysozyme, alkaline phosphatase, antiprotease and respiratory burst activity in serum, blood and intestinal tissue samples were analyzed along with blood differential leukocyte count and tissue histopathology studies. Both humoral and cellular immune responses analyzed were substantially induced or enhanced in the treatment groups in comparison with the control group. The results showed a significant variation in the bio-encapsulation group than the control group and also were comparable to the protection conferred with immersion route immunization under similar conditions. Thus, most of the innate non-specific immune responses are inducible, despite being constitutive of the fish immune system, to exhibit a basal level of protection and a road to better vaccination strategy in Cyprinus carpio L. aquaculture worldwide.

Regulation of Il-10 gene expression by Il-6 via Stat3 in grass carp head kidney leucocytes.

Interleukin (IL)-10 is a critical anti-inflammatory and late cytokine being produced after the proinflammatory mediators while IL-6 is a promptly synthesized cytokine in response to inflammation in mammals. This chronological expression of interleukin (Il)-6 and Il-10 was also found in grass carp head kidney leucocytes (HKLs) treated by heat-killed Aeromonas hydrophila, supporting the possible interplay between grass carp (gc)Il-6 and gcIl-10 in HKLs. Our further findings were in agreement with this hypothesis that recombinant gcIl-6 (rgcIl-6) promptly and transiently increased gcil10 mRNA levels in grass carp HKLs. Moreover, rgcIl-6 enhanced its own mRNA level and this self-enhancement of gcil6 mRNA level could be partially blocked by rgcIl-10. These results collectively suggest that gcIl-10 production stimulated by gcIl-6 may provide a negative feedback to gcIl-6 production. Interestingly, rgcIl-6 significantly decreased gcil10 mRNA levels in grass carp HKLs after the treatment for 12 and 24h in contrast to its enhancement of gcil10 levels after the treatment for 3h. Involvement of Stat3 but not MEK, p38 MAPK or JNK pathway in the increase of gcil10 mRNA levels by rgcIl-6 was revealed by using the signaling pathway inhibitors. This was supported by the fact that rgcIl-6 stimulated Stat3 phosphorylation in grass carp HKLs. Furthermore, rgcIl-6 had no effect on the stability of gcil10 mRNA after the treatment for 3 to 36h while it increased gcil10 promoter activity after the treatment for 24h. Taken these data together, gcIl-6 can stimulate Il-10 production at early stage but subsequently inhibit il10 mRNA expression in grass carp HKLs, shedding light on the dynamic regulation of il10 mRNA expression by Il-6 in fish immune cells.

Characterization and bioactivity of grass carp (Ctenopharyngodon idella) interleukin-21: Inducible production and involvement in inflammatory regulation

In mammals, interleukin 21 (IL-21) is a broad pleiotropic cytokine that plays critical roles in the development of several inflammatory and autoimmune diseases. In fish, functional information of Il-21 is limited, and its role in immune response is largely unknown. In the present study, we cloned a coding sequence of grass carp (Ctenopharyngodon idella) il21 gene (gcil21). To characterize the release patterns and biological activity of gcIl-21, we prepared recombinant gcIl-21 (rgcIl-21) and obtained the polyclonal antibody with gcIl-21 specificity. Western blotting analysis showed that in grass carp head kidney leukocytes (HKLs), gcIl-21 was undetected in culture supernatant of untreated cells but drastically induced by heat-killed Aeromonas hydrophila (A. hydrophila), uncovering the release features of gcIl-21 and its possible involvement in immune response. Subsequent functional experiments revealed that rgcIl-21 did not affect the mRNA expression of grass carp il1b and tgfb, but induced a strong expression of grass carp il10, and to a lesser extent of grass carp tnfa in HKLs, suggesting a dominant effect of gcIl-21 in modulating Il-10 signaling as seen in rainbow trout and mammals. Furthermore, in vivo studies showed that intraperitoneal injection of rgcIl-21 was able to increase the survival rate of grass carp infected with live A. hydrophila, and reduce the pathological responses caused by the same pathogenic bacteria in head kidney and intestine. Taken together, these results for the first time revealed the close relationship of fish Il-21 production and function with inflammatory responses, and highlighted its anti-bacterial and anti-inflammatory ability, thereby providing a new insight into host defense mechanisms in fish.

β-glucan modulates cortisol levels in stressed pacu (Piaractus mesopotamicus) inoculated with heat-killed Aeromonas hydrophila

In this study, we show that β-glucan can modulate cortisol release in fish. We simulated a common situation in aquaculture: the transport of fish followed by contact with an opportunistic pathogen and observed what effect glucan had on the immune and stress response in these conditions. Pacu (Piaractus mesopotamicus) were fed with a diet containing β-glucan (0.1%) for 15 days prior to transport followed by an injection with heat-killed Aeromonas hydrophila. We sampled fish before transport, at arrival and at 3 and 24 h after bacterial injection. β-Glucans are used in aquaculture and have a known immunostimulatory effect, which was observed in this study. The results showed that β-glucan modulated the plasma cortisol levels differently by increasing these levels up to 24 h after transport and preventing the increase caused by bacterial inoculum injection. In addition, β-glucan enhanced the activity of the complement system at 24 h and reduced the monocytes and lymphocytes number in peripheral blood at 3 and 24 h after bacterial inoculation. Our results suggest that β-glucan modulated a bidirectional interaction between the stress and the immune responses. The modulation of cortisol levels and the immunostimulation by β-glucan at different moments in our study suggest the compound has a protective effect by avoiding higher levels of the hormone and improving resistance against bacterial infection in pacu. These results add evidence to support the use of β-glucan as an immunomodulator in the aquaculture industry.

Modulation of stress and innate immune response by corticosteroids in pacu (Piaractus mesopotamicus)

Understanding how stress and corticosteroid modulates the innate immune response is one of the keys to improving productivity and reducing losses in intensive aquaculture. Thus, we investigated the effects of dietary corticosteroids (7 days; long-term exposure) and transport (4 h; short-term stress) on stress and innate immune response in pacu. For this end, fish were fed with diets containing dexamethasone (100 mg kg−1) or hydrocortisone (200 mg kg−1), followed by transport, and then were intraperitoneally inoculated with heat-killed Aeromonas hydrophila or PBS (sham-inoculation). Fish were sampled after a 7-day feeding period, immediately post-transport and 24 h post-transport and inoculation. The dietary treatment of corticosteroids decreased resting cortisol levels by inhibiting the production of cortisol on the hypothalamus pituitary interrenal-axis. Further, both corticosteroids reduced hematocrit, red blood cells, haemoglobin and hemolytic activity of the complement, while they increased glucose levels and serum lysozyme concentrations. The transport increased cortisol and glucose levels and reduced the humoral immune defenses such as serum lysozyme concentration and hemolytic activity of the complement system. Interestingly, the hemolytic activity of the complement system increased sharply in fish fed with corticosteroids immediately post-transport, when they had their HPI-axis partially suppressed by the corticosteroids. This finding suggests a stimulatory effect of the catecholamines released during the transport on the activity of the complement system. Our results are highly valuable to understanding the stress and innate immune responses to long-term exposure to corticosteroids and short-term stress in fish and may provide insights into how corticosteroids modulate the innate immune system.

Modulation of the innate immune responses in the striped snakehead murrel, Channa striata upon experimental infection with live and heat killed Aeromonas hydrophila.

It is well-known that the innate immune mechanisms in fish serve as the first line of defence against wide variety of pathogens. In most of the situations, innate responses get induced and enhanced after the pathogen invasion. It would be interesting to look into the inducibility of various innate immune mechanisms and the level of enhancement after infection with the pathogen. Hence, in the present investigation, modulation of the innate immune responses in the striped snakehead murrel, Channa striata on experimental challenge with either live virulent or heat killed Aeromonas hydrophila at a dose of 1x107 CFU (suspended in 0.2 mL PBS) were measured. Most of the non-specific (both humoral and cellular) immune responses tested were substantially induced or enhanced in both the experimental groups in comparison with the unchallenged control group. Significant increase in the lysozyme, total peroxidase, antiprotease and respiratory burst activities were observed after the pathogen challenge. Thus, most of the innate non-specific immune responses are inducible though they are constitutive of fish immune system exhibiting a basal level of activity even in the absence of pathogen challenge.

A fibrinogen-related protein identified from hepatopancreas of crayfish is a potential pattern recognition receptor.

Fibrinogen-related protein (FREP) family is a large group of proteins containing fibrinogen-like (FBG) domain and plays multiple physiological roles in animals. However, their immune functions in crayfish are not fully explored. In the present study, a novel fibrinogen-like protein (designated as PcFBN1) was identified and characterized from hepatopancreas of red swamp crayfish Procambarus clarkii. The cDNA sequence of PcFBN1 contains an open reading frame (ORF) of 1353 bp encoding a protein of 450 amino acids. Sequence and structural analysis indicated that PcFBN1 contains an FBG domain in C-terminal and a putative signal peptide of 19 amino acids in N-terminal. Semi-quantitative PCR revealed that the main expression of PcFBN1 was observed in hepatopancreas and hemocyte. Temporal expression analysis exhibited that PcFBN1 expression could be significantly induced by heat-killed Aeromonas hydrophila. Tissue distribution and temporal change of PcFBN1 suggested that PcFBN1 may be involved in immune responses of red swamp crayfish. Recombinant PcFBN1 protein binds and agglutinates both gram-negative bacteria Escherichia coli and gram-positive bacteria Micrococcus lysodeikticus. Moreover, binding and agglutination is Ca(2+) dependent. Further analysis indicated that PcFBN1 recognizes some acetyl group-containing substance LPS and PGN. RNAi experiment revealed that PcFBN1 is required for bacterial clearance and survival from A.hydrophila infection. Reduction of PcFBN1 expression significantly decreased the survival and enhanced the number of A.hydrophila in the hemolymph. These results indicated that PcFBN1 plays an important role in the innate immunity of red swamp crayfish as a potential pattern recognition receptor.

Optimisation and standardisation of functional immune assays for striped catfish (Pangasianodon hypophthalmus) to compare their immune response to live and heat killed Aeromonas hydrophila as models of infection and vaccination

Aquaculture production of Pangasianodon hypophthalmus is growing rapidly in South East Asia, especially in Vietnam. As it is a relatively new aquaculture species there are few reports evaluating its immune response to pathogens. Thus, functional assays for P. hypophthalmus were optimised to evaluate both innate and adaptive immune responses, and were then used to examine immune response following stimulation with live and heat-killed Aeromonas hydrophila. These were used as models of infection and vaccination, respectively.Four treatment groups were used, including a control group, a group injected intraperitonally (IP) with adjuvant only, a group injected with heat-killed A. hydrophila (1 × 109 cfu ml−1 mixed with adjuvant), and a group injected with a subclinical dose of live A. hydrophila. Samples were collected at 0, 1, 3, 7, 14 and 21 days post-injection (d.p.i.) to assess their immune response. The results indicated that challenge with live or dead bacteria stimulated the immune response in P. hypophthalmus significantly above the levels observed in control groups with respect to specific antibody titre, plasma lysozyme and peroxidase activity, and phagocytosis by head kidney macrophages at 7 or/and 14 d.p.i. At 21 d.p.i., total and specific antibody (IgM) levels and plasma lysozyme activity in fish injected with either live or dead A. hydrophila were significantly different to the control groups. Differential immune responses were observed between fish injected with either live or dead bacteria, with live A. hydrophila significantly stimulating an increase in WBC counts and plasma peroxidase activity at 3 d.p.i., with the greatest increase in WBC counts noted at 21 d.p.i. and in phagocytosis at 14 d.p.i. By 21 d.p.i. only the macrophages from fish injected with dead A. hydrophila showed significantly stimulation in their respiratory burst activity. This study provides basic information on the immune response in pangasius catfish that can be useful in the health control of this species.

Effect of seeds of Achyranthes aspera on the immune responses and expression of some immune-related genes in carp Catla catla

Evaluation of functional mechanism of immunostimulant in fish is essential for realizing their therapeutic potential. The impact of dietary supplementation of seeds of Achyranthes aspera on the immune system of Catla catla was studied. Catla (37.73 ± 6.7 g) were fed with test diets containing 0.5 (D1) and 1.0% (D2) seeds of A. aspera and control diet (D3) for 50 days. Then fish were immunized with heat-killed Aeromonas hydrophila. Blood and tissue samples were collected after 7 days of immunization. Three fish were sacrificed for each parameter. Respiratory burst activity, bactericidal activity and antibody response were significantly (P < 0.05) higher in test diets fed catla compared to the control one. Nitric oxide synthase and serum lysozyme were significantly (P < 0.05) higher in fish fed with diet D2 compared to the other treatments. There were up-regulations of TNFα, lysozyme c and g gene expressions in kidney and IL-10, lysozyme c and g expressions in hepatopancreas of test diets fed fish compared to the control one. The expression of IL-10 was down-regulated in the kidney of D2 diet fed fish compared to others. In the gill, significantly (P < 0.05) higher expressions of TNFα was found in D1 diet feed fish and lysozyme c and in D2 diet fed fish compared to the other groups. This study showed tissue-specific gene expression pattern in catla. An inverse relationship was found between the expression of TNFα and IL-10 in kidney of catla.

A putative G protein-coupled receptor involved in innate immune defense of Procambarus clarkii against bacterial infection

The immune functions of G protein-coupled receptor (GPCR) were widely investigated in mammals. However, limited researches on immune function of GPCRs were reported in invertebrates. In the present study, the immune functions of HP1R gene, a putative GPCR identified from red swamp crayfish Procambarus clarkii were reported. Expression of HP1R gene was significant up-regulated in response to heat-killed Aeromonas hydrophila challenge. HP1R gene silencing mediated by RNA interference significantly enhanced the susceptibility of red swamp crayfish to A. hydrophila and Vibrio alginolyticus, indicating that HP1R was required for red swamp crayfish to defend against bacterial challenge. In HP1R-silenced crayfish, increased bacterial burden and decreased THC in response to bacterial challenge were observed when compared with control crayfish. No significant difference of proPO gene expression was observed between HP1R-silenced and control crayfish after challenge with heat-killed A. hydrophila. However, PO activity in response to bacterial challenge was significantly reduced in HP1R-silenced crayfish. The results collectively indicated that HP1R was an important immune molecule which was required for red swamp crayfish to defend against bacterial infection.

Involvement of peroxinectin in the defence of red swamp crayfish Procambarus clarkii against pathogenic Aeromonas hydrophila

Cell adhesion factors are important immune components for invertebrate to immobilize, phagocytose or encapsulate invasive microorganisms and foreign particles. In this study, a new cell adhesion factor, peroxinectin (refered as Pcpxin) was isolated from hemocytes of red swamp crayfish ( Procambarus clarkii). The full-length cDNA of Pcpxin was 3014 bp encoding a protein of 819 amino acid residues with a predicted molecular weight of 89.0 kDa and a calculational isoelectric point of 6.93. The putative amino acid sequence contained a peroxidase domain and a signal peptide of 21 amino acid residues, and exhibited high identity to peroxinectin from Pacifastacus leniusculus (85%), Fenneropenaeus chinensis (62%) and Scylla serrata (58%), as well as peroxidase from Camponotus floridanus (40%), Pediculus humanus corporis (39%), and Culex quinquefasciatus (38%). Quantitative real time PCR revealed that mRNA expression of Pcpxin in hemocytes could be inhibited by challenge with heat-killed Aeromonas hydrophila, suggesting that Pcpxin was involved in immune responses to A. hydrophila. RNA interference (RNAi) experiment demonstrated that silencing Pcpxin significantly reduced the survival rate of red swamp crayfishes after challenge with A. hydrophila, which indicated that Pcpxin was important for P. clarkii to survive A. hydrophila infection. Moreover, silencing Pcpxin inhibited the up-regulation of crustin1 and lysozyme expression in response to challenge with heat-killed A. hydrophila. This result suggested that Pcpxin might participate in antibacterial peptide gene expression and thereby might be involved in signal transduction pathway regulating the expression of antibacterial peptide gene.

Changes in biochemical, histological and specific immune parameters in Catla catla (Ham.) by Cynodon dactylon (L.)

Indian major carp, Catla catla (Ham.), was fed with ethanolic extract of Cynodon dactylon (L.) mixed diet with 0.05% (group A), 0.5% (group B) and 5% (group C) extract for 60days with an interval of 10days. Significant (P>0.05) alterations were observed in the haematological and biochemical parameters such as white blood cell counts, red blood cell counts, haemoglobin content, serum glucose, cholesterol, protein, albumin, globulin, albumin/globulin ratio and RNA/DNA ratio in the experimental diet and control fishes. Non-specific immune response was assessed by the anti-protease activity, which was significantly increased in the experimental diet groups. The specific immune response of fish was evaluated by antibody response against heat-killed Aeromonas hydrophila for 28days by using ELISA and bacterial agglutination assay. Aggregation of melanomacrophage centres (MMC) and considerable modifications were observed in the histological analysis of the spleen of experimental diet groups. These results suggested that C. dactylon (L.) could combat the microbial infection by stimulating the immune response in fish.

Febrile response to infection in the American alligator ( Alligator mississippiensis)

Temperature probes were inserted into the stomachs of juvenile American alligators ( Alligator mississippiensis) maintained outdoors at ambient fluctuating temperatures. Internal body temperatures ( T b) were measured every 15 min for two days, and then the alligators were injected with bacterial lipopolysaccharide (LPS), pyrogen-free saline, or left untreated. Alligators injected intraperitoneally with LPS exhibited maximum T bs 2.6 ± 1.1 °C and 3.5 ± 1.2 °C higher than untreated control animals on days one and two after treatment, respectively. T bs for these animals fell to within control ranges by day three postinjection. Similarly, mean preferred body temperatures (MPBTs) were significantly higher for LPS-injected alligators on days one (4.2 ± 1.8 °C) and two (3.5 ± 1.6 °C) after treatment. Intraperitoneal injection of heat-killed Aeromonas hydrophila, a gram-negative bacterium known to infect crocodilians, resulted in a fever while injection of Staphylococcus aureus (gram positive) did not elicit a febrile response. Injection of LPS in alligators maintained indoors in a constant temperature environment resulted in no increase in internal T b. These results indicate that alligators did not exhibit a febrile response in the absence of a thermal gradient, and suggest that febrile responses observed are probably behavioral in nature.

Immune response in the tilapia, Oreochromis mossambicus on exposure to tannery effluent

The objective of the study was to investigate the effect of chronic exposure to sublethal concentrations of tannery effluent (TE) on the specific immune response and nonspecific immunity in tilapia, Oreochromis mossambicus. The effluent from the tannery was collected directly from a chrome-tanning factory situated in Dindigul district, Tamil Nadu, India. Apart from chromium (88.2 ppm), the effluent contained appreciable amount of calcium carbonate and sodium sulphate. Groups of fish (45–50 g) were exposed to 0.0053, 0.053 or 0.53% [0.1%, 1% or 10% LC 50] of TE for 28 days. The specific immune response of fish was assessed by antibody response to heat-killed Aeromonas hydrophila by ELISA and bacterial agglutination assay. Nonspecific immune mechanisms were assessed in terms of serum lysozyme activity, production of intracellular reactive oxygen species (ROS) and reactive nitrogen intermediates (RNI) by peripheral blood leucocytes (PBL). The results indicate that chronic exposure of fish to 0.53% of TE, significantly suppressed antibody response, nonspecific serum lysozyme activity, and ROS and RNI production. Exposure to 0.053% (1% LC 50) of TE also caused a similar suppressive effect though at a lesser degree. In conclusion, the study shows, that exposure to sublethal concentrations of TE, can lead to adverse effects on selected immune reactions in tilapia. Further, these findings may be important in terms of monitoring fish health and risk assessment during periods of fluctuating levels of pollutants in the natural and farm environments.

Effect of Achyranthes aspera on the immunity and survival of Labeo rohita infected with Aeromonas hydrophila

Achyranthes aspera seed was incorporated in the diets (at 0.01%, 0.1% and 0.5%) of Labeo rohita, rohu fingerlings (3.0 ± 0.4 g). After 2 weeks, the fish were immunized with heat-killed Aeromonas hydrophila, and after a further 2 weeks the rohu were experimentally infected with Aeromonas hydrophila (ATCC 49140). After 7 days blood and serum were sampled to determine superoxide anion production, bactericidal activity, lysozyme, serum protein, albumin, globulin, serum glutamate oxaloacetate transaminase (SGOT), serum glutamate pyruvate transaminase (SGPT) and alkaline phosphatase (ALP). Superoxide anion production, serum bactericidal activity, lysozyme, ALP, serum protein, albumin:globulin ratio (A/G) were enhanced in Achyranthes treated groups compared to the control group. SGOT and SGPT levels were elevated in control group, but in Achyranthes treated groups the levels were similar to the uninfected-control group. Higher cumulative mortalities were observed in the control group (77%) up to day-9 after infection. This gradually decreased with increasing dose of Achyranthes, 66% mortality in 0.01% group, 57% mortality in 0.1% group and 28% mortality in 0.5% group. These results indicate that Achyranthes aspera stimulates immunity and increases resistance to infection in L. rohita.

Plasma iron, copper, and zinc in lizard Dipsosaurus dorsalis: effects of bacteria injection.

The effects of inoculation of desert iguanas (Dipsosaurus dorsalis) with heat-killed Aeromonas hydrophila on the plasma concentrations of iron, copper, and zinc, and the total iron-binding capacity have been determined. Plasma iron concentration and total iron-binding capacity fell significantly in inoculated lizards maintained at temperatures corresponding to those selected during fever. Plasma copper concentration did not change in response to inoculation (as determined at 24 h postinjection). Plasma zinc concentration fell in inoculated lizards regardless of whether the lizards were maintained at temperatures corresponding to those selected by afebrile or febrile lizards. There were no effects of varying the zinc concentration (corresponding to those observed in noninfected lizards, infected lizards, or at subnormal levels) on the in vitro growth rate of A. hydrophila. We hypothesize that the reduction in plasma zinc observed in infected mammals, and now in infected lizards, may have an effect (perhaps by some indirect pathway) on the in vivo growth rate of A. hydrophila.