Salmon Erythrocytes Sequester Active Virus Particles in Infectious Salmon Anaemia.

Infectious salmon anaemia virus (ISAV) binds circulating Atlantic salmon erythrocytes, but the relevance of this interaction for the course of infection and development of disease remains unclear. We here characterise ISAV-erythrocyte interactions in experimentally infected Atlantic salmon and show that ISAV-binding to erythrocytes is common and precedes the development of disease. Viral RNA and infective particles were enriched in the cellular fraction of blood. While erythrocyte-associated ISAV remained infectious, erythrocytes dose-dependently limited the infection of cultured cells. Surprisingly, immunostaining of blood smears revealed expression of ISAV proteins in a small fraction of erythrocytes in one of the examined trials, confirming that ISAV can be internalised in this cell type and engage the cellular machinery in transcription and translation. However, viral protein expression in erythrocytes was rare and not required for development of disease and mortality. Furthermore, active transcription of ISAV mRNA was higher in tissues than in blood, supporting the assumption that ISAV replication predominantly takes place in endothelial cells. In conclusion, Atlantic salmon erythrocytes bind ISAV and sequester infective virus particles during infection, but do not appear to significantly contribute to ISAV replication. We discuss the implications of our findings for infection dynamics and pathogenesis of infectious salmon anaemia.

Acclimation to warmer temperature reversibly improves high-temperature hypoxia tolerance in both diploid and triploid brook charr, Salvelinus fontinalis.

Rising temperature leads to reduced oxygen solubility and therefore increases the risk of exposure to harmful hypoxic condition for fish in their natural aquatic environments and in aquaculture. The goal of this study was to determine whether acclimation to warmer temperature can improve high-temperature hypoxia tolerance in fish, using sibling diploid and triploid brook charr as the experimental model. Triploid fish are used for aquaculture and fisheries management because they are sterile, but they are known to have reduced thermal and hypoxia tolerance compared to conventional diploids. Fish were pre-acclimated to either 15 °C (optimum temperature for diploids) or 18 °C and then assessed for high-temperature hypoxia tolerance by rapidly increasing temperature to pre-determined levels (up to 30 °C), holding fish at these temperatures for one hour, and then using compressed nitrogen to drive oxygen out of the water. Hypoxia tolerance was expressed as both the oxygen tension at loss of equilibrium and the time taken to reach this endpoint following the start of the trial. Acclimation to 18 °C improved hypoxia tolerance at high temperatures but this advantage was lost after reacclimation to 15 °C. Although 18 °C acclimation improved the hypoxia tolerance of triploids, it remained inferior to that of diploids under identical test conditions. Somatic energy reserves (estimated as condition factor and hepatosomatic index), cardiac output (relative ventricular mass) and oxygen carrying capacity of the blood (hemoglobin concentration and hematocrit) did not markedly affect high-temperature hypoxia tolerance.

Evidence for a Novel Antiviral Mechanism of Teleost Fish: Serum-Derived Exosomes Inhibit Virus Replication through Incorporating Mx1 Protein.

Exosomes are associated with cancer progression, pregnancy, cardiovascular diseases, central nervous system-related diseases, immune responses and viral pathogenicity. However, study on the role of exosomes in the immune response of teleost fish, especially antiviral immunity, is limited. Herein, serum-derived exosomes from mandarin fish were used to investigate the antiviral effect on the exosomes of teleost fish. Exosomes isolated from mandarin fish serum by ultra-centrifugation were internalized by mandarin fish fry cells and were able to inhibit Infectious spleen and kidney necrosis virus (ISKNV) infection. To further investigate the underlying mechanisms of exosomes in inhibiting ISKNV infection, the protein composition of serum-derived exosomes was analyzed by mass spectrometry. It was found that myxovirus resistance 1 (Mx1) was incorporated by exosomes. Furthermore, the mandarin fish Mx1 protein was proven to be transferred into the recipient cells though exosomes. Our results showed that the serum-derived exosomes from mandarin fish could inhibit ISKNV replication, which suggested an underlying mechanism of the exosome antivirus in that it incorporates Mx1 protein and delivery into recipient cells. This study provided evidence for the important antiviral role of exosomes in the immune system of teleost fish.

Efficacy of ulvan on immune response and immuno-antioxidant gene modulation in Labeo rohita against columnaris disease.

This study reports the effect of ulvan enriched diet on the influence of growth, changes in hemato-biochemical indices, improvement of antioxidant system, enhancement of innate-adaptive immunity and modification of immuno-antioxidant genes expression in Labeo rohita against Flavobacterium columnaris. The weight gain (WG) was significantly high (P > 0.05) in unchallenged normal and challenged fish fed with diets enriched with 25 and 50 mg kg-1 ulvan; the FCR was better (P > 0.05) when fed with 50 mg kg-1 enriched diet. In normal fish fed with or without ulvan supplementation was noted 100% survival rate (SR). In both groups, the red blood cell (RBC) and while blood cell (WBC) counts increased significantly (P > 0.05) when fed with 50 mg kg-1 ulvan diet whereas the hemoglobin (Hb) level increased significantly on being fed with 25 and 50 mg kg-1 ulvan diets. The SOD activity was enhanced significantly in both groups fed with any dose of ulvan diets whereas the MDA and GPx activity increased only with 25 and 50 mg kg-1 ulvan diets. The phagocytic (PC) activity significantly increased with any enriched diet and control diet groups while the respiratory burst (RB) activity increased only with 50 mg kg-1 ulvan diet. The alternate complement pathway (ACP), activity of lysozyme (Lyz), and immunoglobuline M (IgM) were better in both groups fed with 50 mg kg-1 ulvan diet. The SOD and GPx antioxidant gene expression were significantly high in both groups fed with any ulvan diet while the Nrf2 gene expression was high with 50 mg kg-1 ulvan diet. The IL-1ß, TNFα, hepcidin, Lyz, and IgM cytokines or proteins mRNA expression were significant in both groups fed with all ulvan supplement diet whereas the ß-2M expression was significant only with 50 mg kg-1 ulvan diet. The present research indicates that both L. rohita groups fed with 50 mg kg-1 ulvan diet significantly improved growth, antioxidant system, immune defense system, and immuno-antioxidant related gene expression against F. columnaris.

A teleost interleukin-16 is implicated in peripheral blood leukocytes recruitment and anti-bacterial immunity.

Interleukin-16 (IL-16), as a lymphocyte chemoattractant cytokine, plays a crucial role in regulating cellular activities and anti-pathogen immunity. In teleost, the information about the antibacterial effect of IL-16 is scarce. In our study, we examined the immune functions of an IL-16 homologue (CsIL-16) from tongue sole Cynoglossus semilaevis. The CsIL-16 precursor (proCsIL-16) is comprised of 1181 amino acid residues, sharing 21.1%-67.3% identities with IL-16 precursor from invertebrate and vertebrate. The C-terminal proCsIL-16 containing two PDZ domains was designated as mature CsIL-16 which was released into the supernatant of peripheral blood leukocytes (PBLs). CsIL-16 was expressed in various tissues and regulated by bacterial invasion. Recombinant CsIL-16 (rCsIL-16), as a homodimer, was able to bind to the membrane of PBLs and played essential roles in regulating chemotaxis and activation of PBLs, which in vitro inhibited intracellular survival of E. tarda. Under in vivo condition, rCsIL-16 could dramatically regulate the induction of inflammatory genes, and suppress the bacterial dissemination in fish tissues. Collectively, our results reveal that CsIL-16 plays positive roles in antibacterial immunity, and provide insights into the immune function of CsIL-16.

Immune response and protective efficacy of two new adjuvants, Montanide™ ISA 763B VG and Montanide™ GEL02, administered with a Streptococcus agalactiae ghost vaccine in Nile tilapia (Oreochromis niloticus).

Streptococcus agalactiae is one of the most important pathogens infecting tilapia worldwide and causes meningoencephalitis, septicemia and high mortalities with considerable losses. Various types of vaccines have been developed against S. agalactiae infection, such as inactivated vaccines, live attenuated vaccines and subunit vaccines. Bacterial ghosts (BGs) are nonliving, empty cell envelopes and have been reported as novel vaccine candidates. Therefore, the main aims of this study were to develop an S. agalactiae ghost vaccine (SAGV) and to evaluate the immune response and protective effect of SAGV against S. agalactiae with two novel adjuvants, Montanide™ ISA 763B VG and Montanide™ GEL02. Nile tilapia, mean weight 50 g, were divided into four groups as follows; 1) fish injected with PBS as control, 2) fish injected with the SAGV alone; 3) fish injected with the SAGV+Montanide™ ISA 763B VG; and 4) fish injected with SAGV+Montanide™ GEL02. Following vaccination, innate immunity parameters including serum lysozyme, myeloperoxidase, catalase, and bactericidal activity were all significantly enhanced. Moreover, specific serum IgM antibodies were induced and reached their highest level 2-8 weeks post vaccination. Importantly, the relative percent survival of tilapia vaccinated against the SAGV formulated with both adjuvants was 80-93%. Furthermore, the transcription of immune-related genes (IgM, TCRß, IL-1ß, IL-8 and TNFα) were up-regulated in tilapia after vaccination, indicating that both cellular and humoral immune responses were induced by these adjuvanted vaccines. In summary, Montanide™ ISA 763B VG and Montanide™ GEL02 can enhance immunoprotection induced by the SAGV vaccine against streptococcosis, demonstrating that both have value as potential adjuvants of fish vaccines.

Serological responses to koi herpesvirus (KHV) in a non-cyprinid reservoir host.

Koi herpesvirus (KHV) is a highly contagious virus that causes KHV disease (KHVD) inducing high mortality in carp and koi (Cyprinus carpio L.). In the late stage, latency occurs with very low, often non-detectable virus concentrations, which represents a challenge for virus detection. After validation according to OIE recommendations, an antibody ELISA was established to recognize antibodies of C. carpio against KHV infection. In this study, the ELISA was modified to detect anti-KHV antibodies from a non-cyprinid fish. Experimentally infected rainbow trout (Oncorhynchus mykiss) were able to transmit KHV to naïve carp at two different temperatures, demonstrating their potential as a reservoir host. At 20°C, KHVD was induced in carp but not at 15°C. Unexpectedly, rainbow trout developed humoral response against KHV at both temperatures. In contrast to carp, at 15°C trout produced neutralizing antibodies but not at 20°C. While antibodies obtained from infected carp sera reacted in a similar way against all KHV, antibodies from rainbow trout sera reacted differently to the same isolates by ELISA. The data show that even when non-cyprinid fish species are infected with KHV, they can produce antibodies that differ from those observed in carp.

HIGH PREVALENCE OF CIRCULATING ANTIBODIES TO RENIBACTERIUM SALMONINARUM IN SPAWNING ONCORHYNCHUS SPP. FROM LAKE MICHIGAN, USA.

Bacterial kidney disease, caused by Renibacterium salmoninarum, threatens salmonids worldwide. Following devastating mortality episodes in Oncorhynchus spp. in Lake Michigan, US, in the 1980s and infection rates >90%, pathogen prevalence has steadily declined to <5% over three decades in the three state-managed stocks. In this study, we sought to determine if the declining infection rates were associated with heightened circulating antibodies in state-managed Oncorhynchus spp. residing in the Lake Michigan watershed. A single-dilution, indirect enzyme-linked immunosorbent assay (ELISA) was modified to detect circulating antibodies against R. salmoninarum. Baseline values were delineated from naive chinook salmon (Oncorhynchus tshawytscha) and rainbow trout (Oncorhynchus mykiss). The assay was first used to assess primary antibody production over a 4-wk period in chinook salmon experimentally infected with R. salmoninarum. Mean antibody response was detected as early as 2 wk postinfection and continued to increase to the end of the observation period. The modified ELISA was then used to detect antibodies in serum samples collected from feral adult chinook salmon, coho salmon (Oncorhynchus kisutch), and steelhead trout (O. mykiss) returning to spawn at Lake Michigan weirs in 2009 and 2013. Results demonstrated that about 80% of feral Oncorhynchus spp. had measurable titers of circulating antibodies to R. salmoninarum. The relative ease and reasonable costs of this modified ELISA makes it a valuable serosurveillance tool for assessing the humoral immune status of feral salmonid populations.

A multi-biomarker study on Atlantic salmon (Salmo salar L.) affected by the emerging Red Skin Disease in the Baltic Sea.

For half a decade, the Atlantic salmon in the Baltic Sea has been facing severe health issues. Clinical signs like haemorrhage, erosions and ulcerative/necrotic skin conditions in returning adults have been reported from different Swedish rivers. These primary disease signs precede a secondary, terminal fungal infection. As initial investigations of the disease did not provide conclusive answers regarding the pathogenesis, this study was initiated to gain insight into a possible link between this so-called Red Skin Disease and anthropogenic influences. Therefore, returning salmon were caught in rivers along the Swedish coast and different tissues were sampled. The focus was put on the measurements of a battery of biomarkers as well as biochemical and haematological parameters, which were analysed using multivariate statistics. The main findings were a severe osmotic haemodilution, an immune response and an alteration of the carbohydrate metabolism in diseased fish. Furthermore, oxidative stress does not seem to be a likely factor in the pathogenesis. Concluding, certain changes in physiological parameters were shown to be indicative for the disease patterns, while others were ruled out as significant factors. Thus, this study contributes to the understanding of the Red Skin Disease and may act as a hypothesis generator for future studies.

Effect of handling and crowding on the susceptibility of Atlantic salmon (Salmo salar L.) to Lepeophtheirus salmonis (Krøyer) copepodids.

Lepeophtheirus salmonis is an ectoparasite causing economic concerns in Atlantic salmon farming. Salmon lice infestation management methods can be stressful and impact fish welfare. This work investigated the stress effect on the attachment of L. salmonis copepodids to Atlantic salmon through two approaches: (a) handling by netting and air exposure (acute stress), and (b) crowding with restricted surface access in a tank (chronic stress). In the first experiment, we compared the number of attached L. salmonis and cortisol levels between a group of handled salmon and a control group. In the second experiment, a group of densely packed salmon was compared to a control group based on the number of attached copepodids, cortisol levels and neutrophil:lymphocyte ratios. Handled salmon showed significantly higher plasma cortisol levels (p < .001) and more attached copepodids (p = .01) than control salmon. Conversely, the cortisol level and number of attached copepodids were not significantly different between the densely packed and control salmon (p > .05). The neutrophil:lymphocyte ratio was significantly higher (p = .0014) in the densely packed salmon than in the control salmon. Handling salmon increased their risk of infestation by L. salmonis. This has implications for reinfestation rates following delousing treatments in commercial salmon aquaculture, which often involve crowding and handling salmon.

Physiological and molecular responses to thermal stress in red cusk-eel (Genypterus chilensis) juveniles reveals atrophy and oxidative damage in skeletal muscle.

The red cusk-eel (Genypterus chilensis) is a native species with strong potential to support Chilean aquaculture diversification. Environmental stressors, such as temperature, may generate important effects in fish physiology with negative impact. However, no information exists on the effects of thermal stress in Genypterus species or how this stressor affects the skeletal muscle. The present study evaluated for the first time the effect of high temperature stress in red cusk-eel juveniles to determine changes in plasmatic markers of stress (cortisol, glucose and lactate dehydrogenase (LDH)), the transcriptional effect in skeletal muscle genes related to (i) heat shock protein response (hsp60 and hsp70), (ii) muscle atrophy and growth (foxo1, foxo3, fbxo32, murf-1, myod1 and ddit4), and (iii) oxidative stress (cat, sod1 and gpx1), and evaluate the DNA damage (AP sites) and peroxidative damage (lipid peroxidation (HNE proteins)) in this tissue. Thermal stress generates a significant increase in plasmatic levels of cortisol, glucose and LDH activity and induced heat shock protein transcripts in muscle. We also observed an upregulation of atrophy-related genes (foxo1, foxo3 and fbxo32) and a significant modulation of growth-related genes (myod1 and ddit4). Thermal stress induced oxidative stress in skeletal muscle, as represented by the upregulation of antioxidant genes (cat and sod1) and a significant increase in DNA damage and lipid peroxidation. The present study provides the first physiological and molecular information of the effects of thermal stress on skeletal muscle in a Genypterus species, which should be considered in a climate change scenario.

Immune Functions of Erythrocytes in Osteichthyes.

Red blood cells (RBCs)-erythrocytes-of Osteichthyes are primarily known for their involvement in the process of gas exchange and respiration. Currently, physiological properties of RCBs in fish should also include their ability to participate in defense processes as part of the innate and adaptive immune mechanisms. In response to viruses, bacteria, and fungi or recombinant nanoparticles, they can modulate expression of genes responsible for immune reactions, influence activity of leukocytes, and produce cytokines, antimicrobial peptides, and paracrine intercellular signaling molecules. Via the complement system (CR1 receptor) and owing to their phagocytic properties (erythrophagocytosis), RBCs of Osteichthyes can eliminate pathogens. In addition, they are probably involved in the immune response as antigen-presenting cells via major histocompatibility complex class II antigens.

World-wide prevalence of Anisakis larvae in fish and its relationship to human allergic anisakiasis: a systematic review.

The infective stage of Anisakidae nematodes responsible for allergic reactions in humans is found in a variety of edible fish and cephalopods. The identification of geographical regions that are high risk for infected seafood may help prevent allergic reactions in humans. Despite an abundance of published literature which has identified anisakid larvae in an array of edible seafood as well as scattered reports of human allergic anisakiasis, the relationship between the two has not been fully explored. Therefore, a systematic spatio-temporal study was conducted to determine the prevalence of Anisakis spp. in fish from January 2000 to August 2020 firstly to explore the relationship between fish infection and cases of allergic anisakiasis and secondly to use fish infection data to map potential allergic anisakiasis 'hot spots'. A systematic literature search for original English text articles was conducted through search engines, Web of Science, Scopus, PubMed, Science Direct and Google Scholar. Out of 3228 articles which describe anisakid infection in fish, 264 were used for data extraction. Of 904 articles describing allergic anisakiasis, 37 were used for data extraction. A qualitative summary of the extracted data was performed using equal interval method (ArcMap software) in order to compare the global distribution of Anisakis-infected fish. Of the 152-identified fish hosts, five families were most commonly infected with Anisakis spp. These included Lophiidae (86.9%), Trichiuridae (77.05%), Zeidae (70.9%), Merlucciidae (67.8%) and Gadidae (56.8%). The hot spot areas for allergic anisakiasis were North and northeast of Atlantic Ocean, southwest of USA, west of Mexico, south of Chile, east of Argentina, Norway, UK and west of Iceland (confidence 99%). The highest rate of allergic anisakiasis was in Portugal and Norway with the prevalence rate of 18.45-22.50%. Allergologists should consider allergic anisakiasis as a public health issue particularly in high-risk countries where high prevalences in fish have been demonstrated.

Cloning and functional characterisation of natural killer enhancing factor-B (NKEF-B) gene of Labeo rohita: Anti-oxidant and antimicrobial activities of its recombinant protein.

Natural killer enhancing factor (NKEF) of peroxiredoxin family is an important innate immune molecule with having anti-oxidant activity. Although this gene has already been studied in a few fish species, it is yet to be identified and functionally characterised in Indian major carps. In the present study, the complete NKEF-B cDNA of rohu, Labeo rohita was cloned that encoded a putative protein of 197 amino acids. The phylogenetic study showed that L. rohita NKEF-B (LrNKEF-B) is closely related to NKEF-B of Cyprinus carpio and Danio rerio species. Tissue-specific expression of LrNKEF-B gene revealed the highest transcript level in the liver tissue. In the ontogeny study, the highest level of the expression was observed in milt and at 18 h post-development. The expression pattern of this gene was also studied in various pathogen models viz., Gram-negative bacteria (Aeromonas hydrophila), ectoparasite (Argulus siamensis) and a dsRNA viral analogue (poly I:C) in the liver and anterior kidney tissues of L. rohita juveniles. During A. hydrophila infection, the increase in expression of transcripts was observed at 3 h post-infection in both liver (15-fold) and anterior kidney (8-fold). In A. siamensis infection, the expression gradually increased up to 3 d post-infection in the anterior kidney, whereas in liver 3-fold up-regulation was noticed at 12 h post-infection. Similarly, during poly I:C stimulation, up-regulation of NKEF-B transcript was observed in anterior kidney from 1 h to 24 h post-stimulation and down-regulated afterwards whereas, the transcript level increased gradually from 6 h to 15 d post-stimulation in liver tissue. In vitro exposure to concanavalin, A and formalin-killed A. hydrophila upregulated NKEF-B gene expression in anterior kidney and peripheral blood leukocytes of L. rohita, however, down-regulated the same in the splenic leukocytes. A recombinant protein of LrNKEF-B (rLrNKEF-B) of 22 kDa was produced and it showed anti-oxidant activity by protecting supercoiled DNA and reducing insulin disulfide bonds. The minimum bactericidal concentration of this recombinant protein was found to be 4.54 µM against A. hydrophila and Staphylococcus aureus. Interestingly, rLrNKEF-B showed relative percent survival of 72.6 % in A. hydrophila challenged L. rohita, and the survival was found to be associated with a high level of expression of different cytokines, anti-oxidant genes and perforin in the rLrNKEF-B treated L. rohita. An indirect ELISA assay for estimation of NKEF was developed in L. rohita, and the concentrations of NKEF-B increased with time periods post A. hydrophila challenge viz., 0 h (42.56 ng/mL), 12 h (174 ng/mL) and 48 h (370 ng/mL) in rohu serum. Our results suggest a crucial role of LrNKEF-B in innate immunity against biotic stress and oxidative damage and also having antibacterial activity.

The Effects of Ozone on Atlantic Salmon Post-Smolt in Brackish Water-Establishing Welfare Indicators and Thresholds.

Ozone is a strong oxidant, and its use in aquaculture has been shown to improve water quality and fish health. At present, it is predominantly used in freshwater systems due to the high risk of toxic residual oxidant exposure in brackish water and seawater. Here, we report the effects of ozone on Atlantic salmon (Salmo salar) post-smolts (~100 g), in a brackish water (12 ppt) flow-through system. Salmon were exposed to oxidation reduction potential concentrations of 250 mV (control), 280 mV (low), 350 mV (medium), 425 mV (high) and 500 mV (very high). The physiological impacts of ozone were characterized by blood biochemical profiling, histopathologic examination and gene expression analysis in skin and gills. Fish exposed to 425 mV and higher showed ≥33% cumulative mortality in less than 10 days. No significant mortalities were recorded in the remaining groups. The skin surface quality and the thickness of the dermal and epidermal layers were not significantly affected by the treatments. On the other hand, gill histopathology showed the adverse effects of increasing ozone doses and the changes were more pronounced in the group exposed to 350 mV and higher. Cases of gill damages such as necrosis, lamellar fusion and hypertrophy were prevalent in the high and very high groups. Expression profiling of key biomarkers for mucosal health supported the histology results, showing that gills were significantly more affected by higher ozone doses compared to the skin. Increasing ozone doses triggered anti-oxidative stress and inflammatory responses in the gills, where transcript levels of glutathione reductase, copper/zinc superoxide dismutase, interleukin 1ß and interleukin were significantly elevated. Heat shock protein 70 was significantly upregulated in the skin of fish exposed to 350 mV and higher. Bcl-2 associated x protein was the only gene marker that was significantly upregulated by increasing ozone doses in both mucosal tissues. In conclusion, the study revealed that short-term exposure to ozone at concentrations higher than 350 mV in salmon in brackish water resulted in significant health and welfare consequences, including mortality and gill damages. The results of the study will be valuable in developing water treatment protocols for salmon farming.

Quantitative proteomic analysis in serum of Takifugu rubripes infected with Cryptocaryon irritans.

Cryptocaryon irritans can cause cryptocaryonosis (white spot disease) in marine fish but the pathogenesis of the disease is unclear. In this work, we used high-throughput proteomics to identify differentially expressed proteins in the serum of Takifugu rubripes challenged with C. irritans. By using quantitative proteomic assays combined with Tandem Mass Tag-labeled quantitative proteomic analysis, we identified a total of 2088 differentially abundant proteins (1706 proteins were quantified, p < 0.05, fold-change threshold ≥ 2), including 21 up-regulated and 44 down-regulated. Combined with STRING-based functional analysis, we ultimately obtained eight proteins including glucokinase-like, integrin beta-1-like isoform X2, H4, H2A.V, histone H1-like, histone H2AX-like, histone H2B 1/2-like and myosin-9 isoform X1, which could be considered as potential biomarkers for T. rubripes immune responses. Eight proteins that were selected to validate significant differentially expressed genes at the proteomic level were consistent with qPCR at the transcriptomic level. The proteins identified in our work may serve as candidates for elucidating the molecular mechanism of cryptocaryonosis in T. rubripes. Our collective findings could provide new insights into searching for disease-specific targets and biomarkers, which may be effective indicators of C. irritans infection in T. rubripes.

Under stress conditions, pacu Piaractus mesopotamicus modulates the metabolic allostatic load even after Dolops carvalhoi challenge to maintain self-protection mechanisms.

Fish metabolic allostatic dynamics, when animal present physiological modifications that can be strategies to survive, are important for promoting changes to ensure whole body self-protection and survival in chronic states of stress. To determine the impact of sequential stressors on pacu (Piaractus mesopotamicus), fish were subjected to two trials of stressful treatments, administration of exogenous dietary cortisol, and parasite challenge. The first experiment consisted of a two-day acute stress trial and the second, an eight-day chronic stress trial, and after both experiments, fish parasite susceptibility was assessed with the ectoparasite Dolops carvalhoi challenge. Physiological changes in response to acute trial were observed in glycogen, cortisol, glucose, osmolarity, sodium, calcium, chloride, potassium, hematocrit, hemoglobin, red blood cells and mean corpuscular volume, and white blood cell (P < 0.05), whereas response to chronic trial were observed in glycogen, osmolarity, potassium, calcium, chloride, mean corpuscular volume, white blood cell, neutrophil, and lymphocyte (P < 0.05). Acute trials caused physiological changes, however those changes did not induce the consumption of hepatic glycogen. Chronic stress caused physiological changes that induced hepatic glycogen consumption. Under acute trial, stress experience was important to fish to achieve homeostasis after chronic stress. Changes were important to modulate the response to stressor, improve body health status, and overcome the extra stressor with D. carvalhoi challenge. The experiments demonstrate that pacu initiate strategic self-protective metabolic dynamics in acute states of stress that ensure the maintenance of important life processes in front of sequential stressors.

Hematological and immune genes responses in yellow catfish (Pelteobagrus fulvidraco) with septicemia induced by Edwardsiella ictaluri.

Yellow catfish (Pelteobagrus fulvidraco) has been an economically important freshwater species in China because of its good meat quality. In present, the high-density breeding industry has suffered great damage from bacterial infections, in especial, the rapid illness and death of fish caused by bacterial septicemia leads to huge economic losses. Therefore, it is urgent and important to identify pathogenic bacteria and study its pathogenicity. In this study, we isolated a bacterial strain from the yellow catfish with typical septicemia and named it E. 719, then, by morphological observations, regression infection, biochemical identification, 16S rDNA sequence analysis and triple PCR identification, E. 719 was determined to be Edwardsiella ictaluri. Further, we infected yellow catfish with E. ictaluri to study its effects on mortality rate, hematological, histopathological disturbances and expression of immune genes. The mortality results showed that E. ictaluri was highly pathogenic, all infected fish died after 14 days post injection, and the distribution of bacteria in body kidney, spleen, liver, head kidney and brain of fish was continuously detected by measuring the amount of bacteria in the tissues. In addition, the number of red blood cells decreased significantly with the time of infection, while the number of white blood cells and thrombocytes increased. In particular, the number of monocytes and neutrophils increased significantly in the differential leucocyte count (DLC). Histopathologic changes observed by HE staining showed similar results, gill, intestine, spleen and head kidney showed obvious inflammation, bleeding and necrosis. Besides, checking by real time quantitative RT-PCR assays, in both spleen and head kidney tissues which were the major immune organs, mRNA expressions of immune gene IL-1ß, TNF-α, and MR significantly increased in the early and middle stages of infection, which suggested that the infection of E. ictaluri caused a strong immune response in yellow catfish. This study provides a preliminary basis for the diagnosis and treatment of pathophysiology septicemia in yellow catfish induced by E. ictaluri.

Comparison of the blood parameters and histopathology between grass carp infected with a virulent and avirulent isolates of genotype II grass carp reovirus.

Grass carp hemorrhagic disease caused by grass carp reovirus (GCRV) is the most important disease for grass carp aquaculture. Its typical clinical symptom is haemorrhaging, although the mechanism was remained unclear. In this study, we investigated the differences in blood parameters and histopathological features between grass carp infected with a virulent and avirulent isolates of genotype II GCRV. Infection with the virulent isolate resulted in increases in 8 routine blood and 2 serum biochemical parameters (P < 0.05); while 9 routine blood and 5 biochemical parameters were significantly decreased (P < 0.05) compared with fish infected with the avirulent isolate. The majority of these alterations were related to hemorrhage, inflammatory reactions and organic damage. The histopathologic changes were primarily vasodilation and hyperaemia in multiple organs, lymphocyte and macrophage infiltration as well as severe vacuolar degeneration in spleen, kidney and liver. The histopathology changes in fish infected with the avirulent isolate were minimal. These results indicated that the pathogenicity of GCRV was primarily reflected in destruction of the blood circulatory system and parenchymatous organs. This study lays the foundation for further research on the pathogenesis of bleeding caused by GCRV infection and the use of blood parameters and histopathology as tools for disease diagnosis.

Thermal stress causes nuclear and cellular abnormalities of peripheral erythrocytes in Indian major carp, rohu Labeo rohita.

Rise of water temperature as a consequence of global warming is anticipated to affect the physiological activities of fish, especially in tropical regions. In the present experiment, we exposed the Indian major carp, rohu Labeo rohita to three different temperature regimes (30 °C as control and 33 °C and 36 °C) for 60 days and observed the effects of these temperature on: major hemato-biochemical indices (Hemoglobin; Hb, Red blood cell; RBC, White blood cell; WBC and blood glucose levels), erythrocytic nuclear abnormalities (ENA), and erythrocytic cellular abnormalities (ECA) of peripheral erythrocytes along with the formation of differential leucocytes in the blood. Fish were sacrificed at day 7, 15, 30 and 60 after the start of exposure to the temperature regimes. Hb decreased significantly on days 7 and 15 at 36 °C. Throughout the study period, the decrease of RBC and increase of WBC were significant at 36 °C. Blood glucose level increased significantly initially at day 7 but decreased significantly at day 60 at 36 °C. Frequencies of ENA (binucleated, nuclear bud, nuclear bridge, karyopyknosis and notched nuclei) and ECA (twin, fusion, echinocytic, spindle, tear drop and elongated shaped) were significantly increased at the highest temperature (36 °C) at almost all of the sampling days. In the case of differential leucocyte count, high temperature caused a significant increase in the number of neutrophils and a significant decrease in the number of lymphocytes. Overall, these results indicate that chronic exposure to high temperature (36 °C) induces a number of stress responses in rohu and that temperature should be kept below 36 °C in the aquaculture setting to avoid damage to the fish.