Fish and shellfish immunology reports最新文献

Toll-like receptors (TLRs) play key roles in activating immune responses during infection. In this study, we identified TLR genes in Manila clam at the genome-wide level and characterized it into 9 types according to the Ruditapes philippinarum genome annotation, including TLR1 (1–10), TLR2 (1–10), TLR2–2 (1–5), TLR3 (1–3), TLR4 (1–9), TLR5, TLR6 (1–5), TLR7 (1–2), and TLR13 (1–4). The length of TLR proteins varied from 128 to 1257 amino acids. The molecular weights and theoretical isoelectric point (pI) values ranged from 14.63 to 143.32 kDa and 4.47 to 9.45, respectively. TLR genes showed universal expression levels in adductor muscle (AM), mantle (M), foot (F), gill (GI), pipe (P), digestive gland (DG), gonad (GO) and labial palp (L). Transcriptome analysis revealed that the expression level of TLR4, TLR5, TLR7 and TLR13 genes are significantly highly expressed in resistant individuals of Manila clam under Vibrio anguillarum challenge, indicating these TLR genes may play significant roles in response to invading pathogens. The results obtained in this work will provide valuable insights into the immune function of TLR gene in R. philippinarum.

Hemoglobin beta (Hbß) is a heme-binding protein capable of oxygen delivery. The oligopeptides derived from Hbβ in fish mucus are active against a variety of gram-negative bacteria and protozoa. To gain information on the physiological and immunological roles of Hbβ in the mucosal tissues of fish, we analyzed changes in Hbß gene expression levels in the epidermis, gills, and intestine of Japanese flounder, Paralichthys olivaceus, in response to heat stress, Edwardsiella piscicida infection, and trial feeding of immunostimulants, high-concentration ascorbic acid (AsA) or lactoferrin (LF). The results of quantitative real-time PCR showed that expression of the Hbß gene in the gills decreased markedly when exposed to heat stress, whereas that in the epidermis exhibited an increase 3h after infection with E. piscicida. Seven days after starting to feed either immunostimulant, epidermal Hbß gene expression in all AsA or LF dose groups was significantly higher than in the control group. The results of in situ hybridization showed that the abundance and intensity of the stained cells in the epidermis and in the gills were consistent with the expression levels of Hbß gene obtained from the infection and immunosuppressant experiments and the heat stress experiment, respectively. Our results suggest that mucosal Hbβ gene expression is closely related to physiological and immunological status and could be a useful indicator for monitoring condition of fish health.

Interferon Stimulated Gene (ISG)15 is a ubiquitin-like protein that is induced upon viral infections. Our study reports the identification of two homologues of ISG15 in the Asian seabass designated LcISG15A and LcISG15B. The cloned LcISG15A cDNA fragment contained a 474 bp ORF encoding a 157 amino acid protein whereas LcISG15B featured a 498 bp ORF encoding a slightly longer protein of 165 amino acids. Both proteins featured the two tandem ubiquitin-like domains and the C-terminal LRGG motif characteristic of ISG15. The LcISG15B protein has a 10-amino acid C-terminal extension after the LRGG motif. Molecular docking studies revealed that LcISG15A showed more conformational variability of the ubiquitin domains and catalytic function than LcISG15B. The Lates ISG15A and ISG15B genes, reside close in the genome, share the same basic structure with two exons and an intron, but only the second exon encoding the protein. These genes also featured the IFN-stimulatory response elements (ISRE) in the promoter region and ATTTA instability motif in the 3′ UTR region. Leukocyte-rich organs such as the head kidney, heart, spleen, and gill showed higher levels of ISG15A and ISG15B basal expression. Poly (I:C) injection rapidly upregulated the transcription of both the ISG15 genes in these tissues in Lates. In-vivo viral infection by red-spotted grouper nervous necrosis virus also induced upregulation of ISG15 genes in the head kidney, spleen, heart and gill. These findings indicate that the two ISG15 homologues may play a crucial role in innate antiviral immunity and could be used to improve prophylactic strategies and develop species-specific immunological tools for Lates calcarifer.

Edwardsiella tarda is one of the serious bacterial pathogens infecting both cultured and wild catfish urging an immediate need for effective protection strategies. This study assessed the effects of dietary supplementation of Pseudomonas aeruginosa FARP72 at 10⁸ cells/g feed (PA diet) for 30 days on the innate immunity parameters, viz., respiratory oxidative burst (ROB) activity, lysozyme, ceruloplasmin, myeloperoxidase, in-vitro nitric oxide (NO) production in addition to the expression of immune genes encoding interleukin-1β, C3 and transferrin in yellowtail catfish Pangasius pangasius and their resistance to Edwardsiella tarda challenge at a sub-lethal dose of 1.50 × 10⁷ cells/fish. A significant increase in the innate immunity parameters was noted in PA diet-fed catfish on 30 dpf compared to the control. Post E. tarda challenge, the levels of immune parameters increased significantly and peaked at 5 dpi irrespective of feeding to confer protection against E. tarda. Their levels, however, decreased on and from 10 dpi. The results on the expression of immune genes encoding interleukin-1β, C3 and transferrin in the kidney and liver tissue samples of PA diet-fed P. pangasius upon challenge with E. tarda further confirmed the ability of P. aeruginosa to stimulate primary immune organs at the gene level. The effects of feeding P. aeruginosa FARP72 on the immune functions of catfish as examined by the functional immune assays, thus, demonstrating the innate immune responses of catfish that are differentially stimulated by the PA diet. The findings of our study would help evolve management strategies to confer protection against E. tarda infection in commercial catfish aquaculture.

The immunoglobulin (Ig) is a crucial component of adaptive immune system in vertebrates including teleost fish. Here complete cDNA sequence of IgD heavy chain gene from common carp (Cyprinus carpio) was cloned and analyzed. The full-length cDNA of IgD heavy chain gene contained an open reading frame (ORF) of 2460 bp encoding 813 amino acids. According to amino acids sequence, multiple alignment and phylogenetic analysis showed that carp Igs are closely related to those of Cyprinidae fish. Transcriptional expression of IgD as well as IgM, IgZ1 and IgZ2 showed similar expression patterns in different organs, this is, high expression level in systemic immune tissues (ie, head kidney, heart and spleen) and low expression in mucosal tissues (ie, gill, skin and gut). Following viral infection with spring viraemia of carp virus (SVCV), obvious pathological changes in skin, gill and gut mucosa and up-regulated expression of antiviral related genes in skin, gill, gut and spleen were observed, indicating that SVCV successfully infected common carp and activated the systemic and mucosal immune system. Interestingly, IgM showed a significant up-regulation only in systemic tissue (spleen), but not in mucosal tissues (gut, gills and skin), while increased expression of IgZ1 and IgZ2 was found in gut. In contrast, the expression of IgD increased significantly in spleen, gills and skin. These strongly suggest that fish Ig isotypes play different roles in mucosal and systemic immunity during viral infection.

Common carp (Cyprinus carpio); Igs; Spring viraemia of carp virus (SVCV)

Given the intense interest in the use of herbal extracts to improve fish growth, fish health, and disease resistance in fish in culture systems, in this study, we examined the effects of a blend of Guava, Bitter and Neem leaf extracts (GBNL) (i.e., 1:1:1 for GL, BL, and NL respectively) at different inclusion (i.e. 0 GBNL gkg⁻¹, 1 GBNL gkg⁻¹, 3 GBNL gkg⁻¹, 5 GBNL gkg⁻¹, 7 GBNL gkg⁻¹ and 10 GBNL gkg⁻¹) levels on growth, haematology, immunity, liver toxicity and resistance to bacterial co-infections in Nile tilapia. After 8 weeks of feeding, Nile tilapia fed 3 GBNL gkg⁻¹ diets showed significant effects in improving weight gain compared to those fed the control diet. GBNL fed fish showed improved health of fish by stimulating significant increases in levels of White blood cells, Red blood cells, Haemoglobin, and Haematocrit in relation to those fed the control diet. Also, the applications of deferent GBNL levels in Nile tilapia diets showed the potential to upregulate the expression of the immune-related genes heat shock protein 70, chicken type lysozymes, and Beta-defensin, with significant effects shown in fish fed 5GBNL gkg⁻¹ diets in comparison to the control. The results also indicate that GBNL supplementation can decrease mortalities to co-infection of Streptococcus agalactiae and Aeromonas jandaie in Nile tilapia with the lowest mortalities of 13.65% and relative per cent survival of 82.57 % in fish fed 5GBNL gkg⁻¹. Despite the potential of GBNL applications in Nile tilapia, findings of this study indicate fish fed the different concentrations of GBNL, particularly with 7 GBNL gkg⁻¹ can promote the leaching of the liver enzymes: alanine transaminase, aspartate aminotransferase, and alkaline phosphate into the bloodstream which is suggestive of potential liver damage in Nile tilapia. Histological examinations of a cross-section of the liver tissues of fish fed GBNL showed various injuries including hydropic changes, pyknosis nuclei, erythrocytes congestion and vacuolation with the severest seen in those fed 7 GBNL gkg⁻¹. Taking all of the above into consideration, 5GBNL gkg⁻¹ application could improve the health and disease resistance of Nile tilapia; however, prolong use thus after 8 weeks of administration could be injurious to fish liver health.

Elizabethkingia miricola is a highly infectious pathogen, which causes high mortality rate in frog farming. Therefore, it is urgent to develop a rapid and sensitive detection method. In this study, two rapid and specific methods including recombinase polymerase amplification combined with lateral flow dipstick (RPA-LFD) and fluorescent probe-based recombinase polymerase amplification (exo RPA) were established to effectively detect E. miricola, which can accomplish the examination at 38 °C within 30 min. The limiting sensitivity of RPA-LFD and exo RPA (10² copies/μL) was ten-fold higher than that in generic PCR assay. The specificities of the two methods were verified by detecting multiple DNA samples (E. miricola, Staphylococcus aureus, Aeromonas hydrophila, Aeromonas veronii, CyHV-2 and Edwardsiella ictaluri), and the result showed that the single band was displayed in E. miricola DNA only. By tissue bacterial load and qRT-PCR assays, brain is the most sensitive tissue. Random 24 black spotted frog brain samples from farms were tested by generic PCR, basic RPA, RPA-LFD and exo RPA assays, and the results showed that RPA-LFD and exo RPA methods were able to detect E. miricola accurately and rapidly. In summary, the methods of RPA-LFD and exo RPA were able to detect E. miricola conveniently, rapidly, accurately and sensitively. This study provides prospective methods to detect E. miricola infection in frog culture.

Striped catfish, Pangasianodon hypophthalmus was immunized with Biofilm (BF) and Free cell (FC) of Aeromonas hydrophila vaccine at 10¹⁰ CFU g⁻¹ for 20 days and monitored for growth parameters, immune responses and disease resistance up to 60 day post vaccination (dpv). Pangasius catfish in the BF vaccinated group had considerably higher growth and feed utilization than the FC vaccinated and unvaccinated groups (p < 0.05). Biofilm vaccinated group showed a significant increase (p < 0.05) in the mean weight gain (46.91 ± 0.59) than the FC (35.94 ± 0.21) and unvaccinated group (34.92 ± 0.35). The vaccinated fishes were challenged with A. hydrophila at 10⁷ CFU/ml. Significant higher relative percentage survival (RPS) was recorded with BF (84.21 ± 1.49%) compared to that with FC (33.33 ± 1.21%). Polyclonal antibody-based ELISA was used to quantify the antibody titre. BF vaccinated group showed significantly higher antibody titer compared to other treatments (p < 0.05). Moreover, higher haematological parameters recorded in the present study were differentially stimulated by the oral administration of A. hydrophila biofilm vaccine. The mean total protein, albumin, and globulin levels of the BF vaccine groups were significantly higher (p < 0.05) than the mean total protein, albumin, and globulin contents of the unvaccinated group. Furthermore, biochemical stress parameters (SGPT, SGOT) in the vaccinated groups showed an incremental trend in the early days of the experimental period. However, the values were significantly lower (p < 0.05) in the biofilm group on 20 dpv onwards indicating improved health condition. Vaccinated BF fishes showed gut associated lymphoid tissues (GALT) within the laminar propria of mid gut. But in FC group fishes showed less aggregation of lymphoid cells. The unvaccinated control fish had no lymphoid cell aggregation in their intestines. The findings of the current research suggested that biofilm vaccine has the capability to be one of the potential oral vaccines in striped catfish against A. hydrophila infection.

Transient receptor potential vanilloid 4 (TRPV4) is one of the major non-selective cation channel proteins, which plays a crucial role in sensing biotic and abiotic stresses, such as pathogen infection, temperature, mechanical pressure and osmotic pressure changes by regulating Ca²⁺ homeostasis. In the present study, a TRPV4 homologue was identified in Pacific oyster Crassostrea gigas, designated as CgTRPV4. The open reading frame (ORF) of CgTRPV4 was of 2298 bp encoding a putative polypeptide of 765 amino acid residues with three typical ankyrin domains and six conserved transmembrane domains of TRPV4 subfamily proteins, as well as multiple N-glycosylation sites, cAMP- and cGMP-dependent protein kinase phosphorylation sites, protein kinase C phosphorylation sites, casein kinase II phosphorylation sites, and prokaryotic membrane lipoprotein lipid attachment site. The deduced amino acid sequence of CgTRPV4 shared 20.5%-26.2% similarity with TRPV4s from other species. During the early ontogenesis stages of oyster, the mRNA transcripts of CgTRPV4 were detectable in all the stages with the highest expression level in fertilized eggs and the lowest in D-hinged larvae. In adult oyster, the CgTRPV4 mRNA could be detected in all the examined tissues, including gill, hepatopancreas, adductor muscle, labial palp, mantle and haemocyte, with the highest expression level in gill (45.08-fold of that in hepatopancreas, p < 0.05). In immunocytochemical assay, the CgTRPV4 positive signals were distributed in both endoplasmic reticulum and cytoplasmic membrane of oyster haemocytes. The mRNA expression of CgTRPV4 in gill was significantly up-regulated after high temperature stress at 30°C (p < 0.05) and after Vibrio splendidus stimulation (p < 0.05). These results indicated that CgTRPV4 was a classical member of TRPV4 family in oyster, which was induced by either biotic or abiotic stimulations and involved in mediating the stress response of oysters.