Gene characterization and expression pattern of Mx and Viperin genes in Dabry’s sturgeon Acipenser dabryanus

Mx and Viperin are important interferon‐stimulated genes that mediate the antiviral immune response. In this study, we cloned the Mx and viperin genes from Dabry's sturgeon (Acipenser dabryanus). The Mx cDNA sequence contained an open reading frame (ORF) of 1,449 nucleotides, encoding a putative protein of 392 aa, which is significantly shorter than other animal Mx proteins. Although the similarity and identity were low between sturgeon Mx and other animal Mx proteins, sturgeon Mx contains the conserved tripartite GTP binding motif and a dynamin family signature. The sturgeon Mx gene contains eight exons split by seven introns. The sturgeon viperin cDNA sequence contained an ORF of 1,047 bp encoding a putative protein of 349 aa, which is relatively well conserved among species. Sturgeon viperin proteins show 82% similarity with those of Xiphophorus maculatus platyfish and Poecilia formosa Amazon molly. The sturgeon viperin gene has a six exon/five intron structure with the same size of second, third, fourth, and fifth exons between different species. The expression of Mx and viperin was detectable in all tissues examined, with the highest expression in skin for Mx and in peripheral blood for viperin. After mock infection using polyinosine‐polycytidylic acid, Mx and viperin showed significantly upregulated expression in primary spleen leukocytes from 3 hr to 72 hr. Lipopolysaccharide could also induce their expression. These results suggested Mx and Viperin could play a vital antiviral role in the innate immune system of Dabry's sturgeon.     

Identification of interleukin‐16 (IL‐16) and interleukin‐17D (IL‐17D) genes from Dabry's sturgeon (Acipenser dabryanus)

Dabry's sturgeon (Acipenser dabryanus) represents an ancient Actinopterygian lineage that are termed “living fossils”. Many diseases have been found in Dabry's sturgeon. In the present study, genes encoding interleukin (IL)‐16 and IL‐17D in Dabry's sturgeon were identified by RNA‐sequencing. Phylogenetic tree analysis suggested that they clustered together with the corresponding pro‐IL‐16 proteins and IL‐17D proteins from other fish. Sequence analysis revealed that IL‐17D protein was more conserved than pro‐IL‐16. Dabry's sturgeon pro‐IL‐16 contains four putative PDZ domains and do not include signal peptides, while IL‐17D only possesses signal peptides (1–25 aa). The expression patterns of IL‐16 and IL‐17D genes were investigated in Dabry's sturgeon to reveal their functions in disease. The expression level of IL‐16 showed no significant changes in embryos; however, the high expression level of IL‐17D at 0–14 hpf (hours post fertilization) implied the existence of maternal expression in the oocyte and an association with embryonic development. Tissue distribution analysis revealed that IL‐16 and IL‐17D proteins have potential functions in immune and non‐immune tissue compartments. IL‐16 and IL‐17D had different fold changes in primary spleen leukocytes after polyinosinic:polycytidylic acid (poly I:C) and lipopolysaccharide (LPS) administration, which suggested that IL‐16 has a stronger antiviral capability compared with its antibacterial response, and IL‐17D has a stronger antibacterial capability compared with its antiviral response. IL‐16 showed an earlier response to virus compared with IL‐17D, and IL‐17D showed earlier and shorter response to bacteria compared with IL‐16. Our findings suggested the roles of IL‐16 and IL‐17D in Dabry's sturgeon, and provided the theoretical basis for the prevention and control of diseases of Dabry's sturgeon.     

Dietary Macrogard reduces <Emphasis Type="Italic">Aeromonas hydrophila</Emphasis> mortality in tench (<Emphasis Type="Italic">Tinca tinca</Emphasis>) through the activation of cellular and humoral defence mechanisms

Influence of beta-1.3/1.6-glucan (Macrogard) on the innate immunity and protection against Aeromonas hydrophila in tench (Tinca tinca (L.)) was assessed. Macrogard was fed at doses of 0, 0.5, 1 and 2 g kg⁻¹ of pellets for 1 month. The blood, spleen and head kidney from 10 fish of each group were separated and analysed for immunity parameters. Twenty tench from each group were infected with A. hydrophila. Macrogard at doses 1 and 2 g kg⁻¹ of feed significantly (P < 0.05) increased the phagocytic activity of macrophages and proliferative response of mitogens stimulated lymphocytes. The same doses significantly (P < 0.05) increased lysozyme activity and Ig level in serum, compared to the control and dose 0.5 g kg⁻¹ of feed. The challenge test showed that Macrogard reduced mortality of tench after experimental infection (5–35%).     

Expression analysis of cathelicidin and NK‐lysin in Dabry’s sturgeon (Acipenser dabryanus)

Antimicrobial peptides (AMPs) are a conserved component of the innate immune response in many species. In the present study, the cDNA sequences encoding two AMPs (cathelicidin and NK‐lysin, comprising 1,576 and 606 bp, respectively) were cloned from Dabry's sturgeon (Acipenser dabryanus). Phylogenetic analysis demonstrated that the two AMPs were clustered together with homologous protein sequences from other fish. NK‐lysin was highly expressed during early embryonic development, suggesting maternal transmission. Tissue distribution analysis showed that cathelicidin had the highest expression in the liver and NK‐lysin was most abundantly expressed in the spleen. In response to Poly I:C treatment, the expression of cathelicidin was upregulated at 12 and 24 hr post induction (hpi), but downregulated at 72 hpi. NK‐lysin mRNA expression increased after treatment with Poly I:C, reaching a peak at 24 hpi. Lipopolysaccharide treatment also induced the expression of two antimicrobial peptide genes. Lipopolysaccharide treatment significantly upregulated the expression of cathelicidin at 6, 24, and 48 hpi, and upregulated NK‐lysin expression at 6 and 12 hpi. These results suggested that two AMPs could participate in the immune response induced by poly I:C or LPS stimulation.     

Characterization and expression of Megalobrama amblycephala toll‐like receptor 22 involved in the response to Aeromonas hydrophila

The toll‐like receptors (TLR) tlr22 was identified and characterized for the first time in one of the economically most important freshwater fish species in China, Megalobrama amblycephala. The full‐length cDNA (4039 bp) of M. amblycephala tlr22 contains an open reading frame of 2706 bp, encoding a 901 amino‐acid long polypeptide. The putative polypeptide contains 16 leucine‐rich repeat (LRR) motifs, an LRR C‐terminal, a transmembrane region and a cytoplasmic toll–interleukin‐1 receptor (TIR) domain. Phylogenetic analyses revealed that M. amblycephala Tlr22 shared the closest relationship with a grass carp ortholog. tlr22 was constitutively expressed in nine tissues and during 10 developmental stages studied, albeit with varying expression levels. Along with many pathological changes observed after Aeromonas hydrophila bacterium infection, tlr22 and myd88 mRNA were significantly upregulated in blood, head kidney, spleen and intestine, indicating that tlr22 is involved in the immune response. These results provide an insight into tlr22 regulation mechanisms in the innate immune response to bacterial infection.     

Immune response and expression analysis of cathepsin K in goldfish during Aeromonas hydrophila infection

The innate immunity and expression profiles of cathepsins D were determined in the goldfish (Carassius auratus) tissues after challenge with a fish pathogen Aeromonas hydrophila. The innate immunity of reactive oxygen species (ROS) and reactive nitrogen species (RNS) were determined by peripheral blood leucocytes. Blood and tissue samples of the muscle, gills, liver, kidney, heart, spleen, and intestine were sampled at 1, 3, 6 and 12 h post-infection for cathepsin D expression by semi-quantitative RT-PCR. The ROS and RNS production did not significantly increase at 1 h post-challenged goldfish. However, the ROS and RNS production was significantly increased after 3 h post-challenged fish compared to the control. The cathepsin D expression was found very low in muscle and kidney of the control fish, other tissues was not found the expression. A similar pattern was found in goldfish at 1 h post-challenge with A. hydrophila. However, at 3 h post-challenge goldfish, the cathepsin D expression was high only in the heart. At 6 h post-challenge goldfish, the cathepsin D expression was seen high all the tissues, except in the spleen. However, the expression was decreased at 12 h post-infection samples. This result was suggested that the goldfish infected with A. hydrophila decreased the innate immunity level in peripheral blood and expressed the cathepsin D in tissues.     

Identification of Omp38 by immunoproteomic analysis and evaluation as a potential vaccine antigen against Aeromonas hydrophila in Chinese breams

Aeromonas hydrophila is a fish pathogen causing systemic infections in aquatic environments, and determining its antigenic proteins is important for vaccine development to reduce economic losses in aquaculture worldwide. Here, an immunoproteomic approach was used to identify immunogenic outer membrane proteins (OMPs) of the Chinese vaccine strain J-1 using convalescent sera from Chinese breams. Seven unique immunogenic proteins were identified by two-dimensional (2-D) electrophoresis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-TOF-MS). One protein of interest, Omp38, was expressed, and its immunogenicity and protective efficacy were evaluated in Chinese breams. The two groups of fish immunized with the inactivated vaccine and recombinant Omp38 protein showed significant serum IgM antibody levels after vaccination, compared with the fish injected with PBS buffer. In addition, the superoxide dismutase (SOD) activity, lysozyme (LSZ) activity and phagocytosis activity of head kidney lymphocytes of immunized groups were significantly higher than those of the control. The fish receiving inactivated vaccine and recombinant Omp38 protein developed a protective response to a live A. hydrophila challenge 45 days post-immunization, as demonstrated by increased survival of vaccinated fish over the control and by decreased histological alterations in vaccinated fish. Furthermore, protective effect was better in Omp38 group than in the inactivated vaccine group. These results suggest that the recombinant Omp38 protein could effectively stimulate both specific and non-specific immune responses and protect against A. hydrophila infection. Therefore, Omp38 may be developed as a potential vaccine candidate against A. hydrophila infection.     

Isolation and Identification of a Novel Inducible Antibacterial Peptide from the Skin Mucus of Japanese Eel, <Emphasis Type="Italic">Anguilla japonica</Emphasis>

In this study, acetone extracts and acidic extracts were prepared from skin mucus, gill, kidney, liver and spleen of the Japanese eel, Anguilla japonica, and they exhibited different levels of antibacterial activities against three strains of Gram-negative bacteria, Edwardsiella tarda, Aeromonas hydrophila, Aeromonas sp. and one Gram-positive bacterium Micrococcus leteus. The mucus was chosen as the source of antibacterial peptide for further purification of antibacterial peptides. Following the intraperitoneal injection of A. hydrophila, one of the main pathogenic bacteria of Japanese eel and many other fish, a peptide was purified from acetic acid extraction of the skin mucus, by using cationic exchange liquid chromatography and reverse-phase high-performance liquid chromatography (RP-HPLC). The isolated antibacterial peptide, named as AJN-10, exhibited antibacterial activity against A. hydrophila. The AJN-10 is a heat-tolerant and hydrophilic peptide. The molecular weight of this peptide is 6,044.28 Da, as determined by matrix-assisted laser desorption ionisation time of flight mass spectrometry. The 20 N-terminal amino acid sequences were clarified by Edman degradation, and based on results of homology search by BLAST analysis of the 20 N-terminal sequences, the AJN-10 showed little similarity to other proteins in databases.     

Effects of intraperitoneal injection of cortisol on non‐specific immune functions of Ctenopharyngodon idella

After grass carps Ctenopharyngodon idellus were injected with cortisol, with (CBC) and without (C) a cocoa butter carrier, the effects of both slowly and rapidly acting exogenous cortisol on their non‐specific immune functions were investigated. On the one hand, after injection with CBC, the cortisol concentration and lysozyme activity in fish serum were enhanced and were sustained at high levels for a long period (30 days). The killing activity in the serum declined with time, and phagocytosis of head kidney macrophages diminished significantly (P < 0·05 or P < 0·01). The leukocrit values in the high dose group (31·8 mg cortisol fish^?1) increased over time, however, with the maximum average being 5·6% at day 30. The spleen mass index in the high dose group was 0·93 × 10^?3 after 30 days, notably lower (P < 0·05) than that in the control group. In addition, a decrease in resistance to Aeromonas hydrophila infection in cortisol‐treated fish was shown, with the final cumulative mortalities being 54·5 and 66·7% in the low and high dose groups, respectively. On the other hand, there was a decrease in both serum cortisol concentration and lysozyme activity of the experimental fish within 2 weeks after injection with C, where plasma bactericidal activities in the high dose group (31·8 mg cortisol fish^?1) were remarkably lower (P < 0·01) than those in the control group at each sampling, but were increased slightly over time. The results of which were different from those in the CBC trial. Phagocytic activity of head kidney macrophages and spleen mass index decreased significantly (P < 0·05), while there were increases in leukocrit value and cumulative mortality due to A. hydrophila. The results of which were similar to those in the CBC trial. This study indicated that the injection of cortisol depressed the non‐specific immune functions of the grass carp and increased its susceptibility to disease.     

Leishmania donovani infection differentially regulates small G‐proteins

Leishmania is a protozoan parasite that resides and replicates in macrophages and causes leishmaniasis. The parasite alters the signaling cascade in host macrophages and evades the host machinery. Small G‐proteins are GTPases, grouped in 5 different families that play a crucial role in the regulation of cell proliferation, cell survival, apoptosis, intracellular trafficking, and transport. In particular, the Ras family of small G‐proteins has been identified to play a significant role in the cellular functions mentioned before. Here, we studied the differential expression of the most important small G‐proteins during Leishmania infection. We found major changes in the expression of different isoforms of Ras, mainly in N‐Ras. We observed that Leishmania donovani infection led to enhanced N‐Ras expression, whereas it inhibited K‐Ras and H‐Ras expression. Furthermore, an active N‐Ras pull‐down assay showed enhanced N‐Ras activity. L donovani infection also increased extracellular signal–regulated kinase 1/2 phosphorylation and simultaneously decreased p38 phosphorylation. In contrast, pharmacological inhibition of Ras led to reduction in the phosphorylation of extracellular signal–regulated kinase 1/2 and enhanced the phosphorylation of p38 in Leishmania‐infected cells, which could lead to increased interleukin‐12 expression and decreased interleukin‐10 expression. Indeed, farnesylthiosalicyclic acid (a Ras inhibitor), when used at the effective level in L donovani–infected macrophages, reduced amastigotes in the host macrophages. Thus, upregulated N‐Ras expression during L donovani infection could be a novel immune evasion strategy of Leishmania and would be a potential target for antileishmanial immunotherapy.     

Neutrophil activity affects Oreochromis mossambicus (Peters, 1852) antibody production against heat‐killed Aeromonas hydrophila vaccine

Aeromonas hydrophila is one of the important and most common pathogens of warm water fish. Prophylactic and therapeutic measures against A. hydrophila infection are essential to prevent loss of fish production in aquaculture. A heat‐killed vaccine was developed against three strains of A. hydrophila, namely O21, O26 and O28, and analysed for their comparative immunogenicity in Oreochromis mossambicus (Peters, 1852). Studied were the neutrophil activity and specific antibody response of the host against the vaccines, which showed that neutrophil activity was highest for the heat‐killed O21, but that the heat‐killed O28 produced the highest antibody titres. The antibody cross‐reactivity tests indicated that the antibody raised against O28 was pan‐reactive whereas it was less cross‐reactive in O21. Thus strain O28 may be used as a vaccine candidate for a pan‐protection of fish from various strains of A. hydrophila infections. However, further rigorous studies with different fish species and bacterial strains are needed to confirm these results.     

Significance and suitability of <Emphasis Type="Italic">Aeromonas hydrophila</Emphasis> vs. fecal coliforms in assessing microbiological water quality

We examined the significance and suitability of Aeromonas hydrophila versus fecal coliforms in assessing microbiological water quality. For this, we used the membrane filtration method to simultaneously estimate the abundance level of A. hydrophila and fecal coliforms in waters from the Mfoundi river watershed at Yaoundé, and compared how fluctuations in A. hydrophila abundance matched those observed with fecal coliforms index as an indicator of water quality in the system under study. Our results revealed that waters were not safe according to the standards for water quality established by the Word Health Organization (WHO). They also indicated the prevalence of A. hydrophila as compared to fecal coliforms, and suggested that water from the Mfoundi River and its tributaries could be classified as hypereutrophic based on the density of Aeromonas. Moreover, the spatial distribution of fecal coliforms and A. hydrophila exhibited similar trends within the different water bodies investigated, suggesting that A. hydrophila can be used as indicator of water quality in highly polluted waters. We concluded that waters from the Mfoundi River watershed at Yaoundé represent a great potential risk of infection for users, and foresee that the next challenge will be to determine, among other factors, the physico-chemical factors influencing the observed spatial distribution.     

Laboratory study of the susceptibility of hatchery reared Atlantic sturgeon (Acipenser oxyrinchus Mitchill, 1815) larvae to predation

The aim of the study was to determine the susceptibility to predation of Atlantic sturgeon larvae (Acipenser oxyrinchus) reared under traditional hatchery conditions. Experiments were conducted to determine whether predators would prey on Atlantic sturgeon if alternative prey was available and if the presence of substrate on the tank bottom impacted the number of Atlantic sturgeon consumed. European perch (Perca fluviatilis) was used as the predator, and the alternative prey were three‐spined stickleback (Gasterosteus aculeatus) or gudgeon (Gobio gobio). The predators and alternative prey were obtained from the wild. The mortality of sturgeon (n = 10) and alternative prey (n = 10) caused by four predators was recorded during 15 min trials. Trials with three‐spined stickleback and gudgeon as alternative prey were performed separately. Each experimental trial was repeated five times. The predators consumed significantly more Atlantic sturgeon than alternative prey in both the experimental setups when the bottom of the tank was covered with gravel and stone substrate and when there was no substrate. In trials with three‐spined stickleback the mortality of Atlantic sturgeon in both experimental setups was 94 ± 8.94%, while that of three‐spined stickleback in the setup with substrate was 20 ± 19.23%, and without substrate it was 22 ± 10.00%. European perch also consumed more Atlantic sturgeon than they did gudgeon, and the mean Atlantic sturgeon mortality in the experimental setup with substrate was 94 ± 5.48%, while for gudgeon it was 48 ± 8.37%. In the experimental setup without substrate the predators also consumed substantially more Atlantic sturgeon than gudgeon, with a mean Atlantic sturgeon mortality of 94 ± 8.94%, while for gudgeon it was 76 ± 5.48%. The study indicated that hatchery reared Atlantic sturgeon larvae are susceptible to predation by European perch. Predation could impact the survival of juvenile Atlantic sturgeon in the natural environment, and it could be one of the factors that is impeding the restoration of this species in the Baltic Sea basin.     

Updated assessment of hatchery‐reared pallid sturgeon (Forbes & Richardson, 1905) survival in the lower Missouri River

As pallid sturgeon, Scaphirhynchus albus (Forbes & Richardson, 1905), natural reproduction and recruitment remains very minimal in the lower Missouri River from Gavins Point Dam (river kilometer [rkm] 1305.2) to the confluence with the Mississippi River (rkm 0.0), hatchery supplementation and river‐wide monitoring efforts have continued. Annual survival estimates of hatchery‐reared pallid sturgeon stocked in the lower Missouri River were previously estimated during 1994–2008. Low recapture rates prior to 2006 limited the data available to estimate survival, which resulted in considerable uncertainty for the estimate of annual survival of age‐1 fish. Therefore, the objective was to provide more precise estimates of annual survival of pallid sturgeon using five additional years of stocking and sampling. The Cormack‐Jolly‐Seber model structure provided in program MARK was used to estimate the age‐specific survival estimates. Over 135 000 hatchery‐reared pallid sturgeon were released during 1994–2011 and recaptured at a rate of 1.9%, whereby estimates for the annual survival of age‐0 (Ø = 0.048) and >age‐1 (Ø = 0.931) were similar to those previously reported, but the age‐1 (Ø = 0.403) survival estimate was 52% lower. Post hoc analysis using time‐specific survival estimates indicated lower survival for age‐1 fish post‐2003 year classes, relative to the pre‐2002 year classes. An analysis confirms that hatchery‐reared pallid sturgeon continue to survive in the wild. However, low survival during the first 2 years of life is a management concern as efforts are aimed at maximizing genetic diversity and population growth. A follow‐up analysis also demonstrated the variability of capture rates and survival over time, which reinforces the need to continue to monitor and evaluate mark‐recapture data. The mark‐recapture efforts have provided demographic parameter estimates that remain a critical component for species recovery as these data are incorporated into population models.     

A Streptococcus iniae DNA vaccine delivered by a live attenuated Edwardsiella tarda via natural infection induces cross‐genus protection

Edwardsiella tarda and Streptococcus iniae are important fish pathogens. We have reported previously a live E. tarda vaccine based on the attenuated strain TX5RM and a S. iniae DNA vaccine based on the antigen Sia10. In this study, we examined the possibility of constructing a cross‐genus vaccine by taking advantage of the residual infectivity of TX5RM and using it as a carrier host for the natural delivery of a S. iniae DNA vaccine. For this purpose, the recombinant TX5RM, TX5RMS10, was created, which harbours and retains stably the DNA vaccine plasmid pCS10 that expresses Sia10. When flounder were vaccinated with TX5RMS10 via oral and immersion routes, TX5RMS10 was detected in multiple tissues within 12–14 days postvaccination (p.v.). At 7 and 14 days p.v., expression of the DNA vaccine was detected in spleen, kidney and liver. Following E. tarda and S. iniae challenge at one and 2 months p.v., the vaccinated fish exhibited relative per cent survival rates of 69–83%. Immunological analysis indicated that TX5RMS10‐vaccinated fish produced specific serum antibodies and exhibited enhanced expression of a wide range of immune genes.     

Effects of dietary zearalenone on the serum biochemistry,hepatic and intestinal histology,and intestinal microbiota of juvenile Dabry′s sturgeon (Acipenser dabryanus)

Zearalenone (ZEN) is a common contaminant in animal feed, but the adverse effects of ZEN on the intestinal microbiota of fish have not yet been investigated. To reveal the effects of ZEN on serum biochemistry, hepatic and intestinal histology, and intestinal microbiota of fish, Dabry′s sturgeon (Acipenser dabryanus) received feed containing 1030 μg kg⁻¹ ZEN (ZEN-treated group) for 7 weeks and were compared to a control feed that have not been fortified with ZEN (control group). The results showed that dietary supplementation with ZEN did not significantly affect the serum contents of alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, high-density lipoprotein cholesterol (HDLC) and estradiol (E₂; p > .05). The serum contents of total protein, albumin, triglycerides, total cholesterol and low-density lipoprotein cholesterol in the ZEN-treated group were significant lower than the control group (p < .05). The serum contents of glucose in the ZEN-treated group was significant higher than the control group (p < .05). Intestinal histology was normal in the ZEN-treated group with comparsion to the control group. Compared to the control group, the appearance of nuclear pyknosis and vacuoles in the hepatic cell in the ZEN-treated group. The α-diversity index (Chao 1, Faith pd and Shannon diversity index) significant increased in the ZEN-treated group compared to the control group (p < .05). Simpson diversity index was not affected by the dietary ZEN-treated (p > .05). Principal coordinates analysis (PCA) showed different clustering of prokaryotic communities in the ZEN-treated group compared to the control group. Further Anosim (Analysis of similarities) found that significnat differences in species community structure composition (R > .44) between the control group and the ZEN-treated group (p < .05). The number of operational taxonomic units (OTUs) was decreased after Dabry's sturgeon were fed diets supplemented with ZEN. At the genus level, the differences in the relative abundances of the first 20 main microbiota genera were considerable. In summary, this study suggests that dietary containing 1030 μg kg⁻¹ ZEN may be interfere with physiological parameters, and also affect the intestinal microbiota α-diversity, the numbers of OTUs and the microbiota compostion at the genus level of the juvenile Dabry′s sturgeon.