Infection dynamic of Micropterus salmoides rhabdovirus and response analysis of largemouth bass after immersion infection.

Largemouth bass (Micropterus salmoides) is an important economic freshwater aquaculture fish originating from North America. However, the frequent outbreaks of Micropterus salmoides rhabdovirus (MSRV) have seriously limited the healthy development of Micropterus salmoides farming industry. In the present study, a strain of MSRV was isolated and identified from infected largemouth bass by PCR, transmission electron micrograph observation and genome sequences analysis, and tentatively named MSRV-HZ01 strain. Phylogenetic analyses showed that the MSRV-HZ01 presented the highest similarity to MSRV-2021, followed by MSRV-FJ985 and MSRV-YH01. The various tissues of juvenile largemouth bass exhibited significant pathological damage following MSRV-HZ01 immersion infection, and the mortality reached 90%. We also found that intestine was the key organ for MSRV to enter the fish body initially by dynamic analysis of viral infection, and the head kidney was the susceptible tissue of virus. Moreover, the MSRV was also transferred to the external mucosal tissue in later stage of viral infection to achieve horizontal transmission. In addition, the genes of IFN γ and IFN I-C were significantly up-regulated after MSRV infection to exert antiviral functions. The genes of cGAS and Sting might play an important role in the regulation of interferon expression. In conclusion, we investigated the virus infection dynamics and fish response following MSRV immersion infection, which would promote our understanding of the interaction between MSRV and largemouth bass under natural infection.

An Evaluation of Laminarin Additive in the Diets of Juvenile Largemouth Bass (Micropterus salmoides): Growth, Antioxidant Capacity, Immune Response and Intestinal Microbiota.

A 28 day feeding trial was conducted to investigate the growth performance, immune response and intestinal microbiota of laminarin (LAM) supplemented diets in juvenile largemouth bass (Micropterus salmoides). Four hundred and eighty fish (initial average weight: 0.72 ± 0.04 g) were randomly divided into four groups (40 fish per tank with three replicates in each group) Four diets were prepared with LAM supplementation at the doses of 0 (control), 5 g Kg-1 (LL), 10 g Kg-1 (ML) and 15 g Kg-1 (HL), respectively. No significant difference in the specific growth rate (SGR) and hepatosomatic index (HSI) was observed in fish among the four groups, or in the lipid and ash content of fish flesh. In addition, fish in the LL group exhibited much higher antioxidant capacity (p < 0.05), while the diets with the inclusion of 5 and 10 g Kg-1 LAM remarkably decreased the antioxidant capacity of fish (p > 0.05). Dietary LAM at the dose of 5 g Kg-1 inhibited the transcription of interleukin-1ß (il-1ß) and tumor necrosis factor-α (tnf-α), while promoting the expression of transforming growth factor-ß (tgf-ß) in fish intestine. Moreover, the beneficial intestinal bacteria Bacteroide, Comamonas and Mycoplasma abundance significantly increased in fish from the LL group, while the content of opportunistic pathogens Plesiomonas, Aeromonas and Brevinema in fish of the HL group was substantially higher than the control group. Overall, the appropriate dose of supplemented LAM in the diet was 5 g Kg-1, while an excessive supplementation of LAM in the diet led to microbial community instability in largemouth bass.

The role of TNF-α in the phagocytosis of largemouth bass (Micropterus salmoides) leukocytes.

Phagocytosis is an important innate immune process in which immune cells recognize, ingest and eliminate pathogens. Largemouth bass (Micropterus salmoides) has become an important economic farmed fish in many regions, while few studies has focused on phagocytosis of its leucocytes. In present study, largemouth bass peripheral blood leucocytes were separated using Percoll gradient to establish the phagocytic function. Flow cytometric analysis showed that largemouth bass leukocytes exhibited the phagocytic capacity to fluoresbrite microspheres and Aeromonas hydrophila, where higher phagocytic capacity to A. hydrophila were observed in granulocytes/monocytes than that of lymphocytes. The leukocytes engulfing fluoresbrite microspheres and A. hydrophila were also observed by fluorescence microscopy. Besides, manygenes associated with phagocytosis and TNF-α in leukocytes were up-regulated following A. hydrophila stimulation. Subsequently, the largemouth bass TNF-α was recombinantly expressed to investigate its role in regulating phagocytosis. The results showed that TNF-α in largemouth bass could significantly enhance the phagocytic ability of granulocytes/monocytes to A. hydrophila, but not lymphocytes. Moreover, we also found that TNF-α could not only significantly increase the ROS activity of granulocytes/monocytes, but also had the function of inducing its apoptosis. These results demonstrated that granulocytes/monocytes play more important role in phagocytosis, meanwhile, TNF-α has the function of enhancing the phagocytic ability of granulocytes/monocytes in largemouth bass.

Protective efficacy evaluation of immunogenic protein AHA_3793 of Aeromonas hydrophila as vaccine candidate for largemouth bass Micropterus salmoides.

Aeromonas hydrophila is a Gram-negative pathogen that can infect various fish, including largemouth bass (Micropterus salmoides), which have caused huge economic losses. In present study, largemouth bass anti-A. hydrophila antibodies were produced, then a highly immunogenic outer membrane proteins, AHA_3793, was identified by combined western blotting and mass spectrometry analysis. Moreover, AHA_3793 was expressed, and its immunogenicity was further verified by western blotting. Subsequently, the protective efficacy of AHA_3793 were evaluated in largemouth bass. The results showed that rAHA_3793 could produce a relative percentage survival (RPS) of 61.76% for largemouth bass against A. hydrophila challenge. ELISA analysis showed the specific serum antibodies of largemouth bass against rAHA_3793 and A. hydrophila in vaccinated group in weeks 4 and 5 after immunization were significantly higher than those in control group, which suggested that rAHA_3793 induced production of specific serum antibodies against rAHA_3793 and A. hydrophila. The qRT-PCR analysis showed that expressions of CD4-2 and MHC IIα were also significantly up-regulated after immunization. These results collectively demonstrated that rAHA_3793 could induce a strong humoral immune response of largemouth bass, and then produce high immune protection effects against A. hydrophila infection.

C-type lectin with a QPN motif from swimming crab Portunus trituberculatus displays broad nonself-recognition ability and functions as an opsonin.

In the immune system, C-type lectins, as pattern recognition receptors, have an important function. Carbohydrate-recognition domains (CRDs) endow C-type lectins with the function of recognizing and scavenging non-self factors. In the present study, a new C-type lectin (designated as PtCTL-9 according to the order of discovery) from swimming crab (Portunus trituberculatus) was characterized. QPN (Gln-Pro-Asn) and FHS (Phe-His-Ser) were identified as the key motifs that determine carbohydrate binding. Motif QPN was mutated to QPD (Gln-Pro-Asp) (M1) and EPN (Glu-Pro-Asn) (M2) to study its immune function and for comparative analysis. The results showed that PtCTL-9 displayed broad non-self immunity. PtCTL-9 could also function as an opsonin to promote phagocytosis and the in vitro encapsulation of hemocytes. These results indicated that PtCTL-9 has an extensive nonself-recognition ability, regulates pathogen clearance, and its QPN motif is important in PtCTL-9's immune function.

Molecular cloning and binding analysis of polymeric immunoglobulin receptor in largemouth bass (Micropterus salmoides).

The polymeric immunoglobulin receptor (pIgR) is an important molecule in the mucosal immunity of teleosts. Previous studies have shown that pIgR can bind and transport polymeric immunoglobulins (pIgs), but few studies have focused on the binding of teleost pIgR to bacteria. In this study, we identified a gene encoding pIgR in largemouth bass (Micropterus salmoides). The pIgR gene contained two Ig-like domains (ILDs), which were homologous to ILD1 and ILD5 of mammalian pIgR. Our results showed that largemouth bass pIgR-ILD could combine with IgM. Moreover, we also found that largemouth bass pIgR-ILD could bind to Aeromonas hydrophila and Micrococcus luteus. Further analysis showed that largemouth bass pIgR-ILD could also combine with lipopolysaccharide (LPS), peptidoglycan (PGN) and various saccharides, and reduced binding to bacteria was observed with LPS and PGN treatment, indicating that largemouth bass pIgR could bind to bacteria to prevent infection and that saccharide binding is an important interaction mechanism between pIgR and bacteria. These results collectively demonstrated that largemouth bass pIgR not only combines with IgM but also binds to bacteria by various saccharides.

Effect of biological additives on Japanese eel (Anguilla japonica) growth performance, digestive enzymes activity and immunology.

Japanese eel (Anguilla japonica) has become a commercially important fish species all over the world. High-density aquaculture has led to congestion and contributed to bacterial infection outbreaks that have caused high mortality. Therefore a 56-days feeding trial was conducted to determine the effects of dietary Bacillus amyloliquefaciens (GB-9) and Yarrowia lipolytica lipase2 (YLL2) on growth performance, digestive enzymes activity, innate immunity and resistance to pathogens of A. japonica. Fish growth performance was significantly affected by dietary YLL2 supplementation but not by GB-9. Fish fed diets with YLL2 at 2.0 g/kg diet in combination of high and low levels of GB-9 (5.0 g/kg and 2.0 g/kg) produced the highest growth. For digestive enzyme, lipase and trypsin activities was promoted by dietary containing YLL2, while amylase activities was increased by dietary containing YLL2, GB-9 single or combination. For innate immunity, the mucus lysozyme activity, leukocytes phagocytosis activity and reactive oxygen species level of skin, peroxidase and lysozyme activity of serum were enhanced in fish fed with GB-9 compared to those in control group (p < 0.05). The highest resistance to Vibrio anguillarum and Aeromonas hydrophila was determined in fish fed with 5.0 g kg-1 GB-9 + 2.0 g/kg YLL2. This study demonstrated that GB-9 and YLL2 enhanced non-specific immune defense system of A. japonica, providing them with higher resistance to pathogens. The present results suggested that the combination of these supplements could be considered as potential biological additives for aquaculture farmed fish.

Effects of Bacillus amyloliquefaciens and Yarrowia lipolytica lipase 2 on immunology and growth performance of Hybrid sturgeon.

A 12-weeks feeding trial was performed to investigate the possible effects of supplementation of Hybrid sturgeon diet with Bacillus amyloliquefaciens (GB-9) and Yarrowia lipolytica lipase2 (YLL2) single or combined on immune response and growth performance of Hybrid sturgeon (Acipenser schrenkii ♂and Acipenser baeri ♀). For this aim, Hybrid sturgeons were fed with four experimental diets namely: Diet 1 (0-control), Diet 2 (5.0 g/kg GB-9), Diet 3 (4.0 g/kg YLL2), and Diet 4 (5.0 g/kg GB-9 + 4.0 g/kg YLL2), respectively. After fed with varied diets, growth performance, mucosal immune response, leukocytes immune response and serum immunological response were measured. The results indicated that supplementations of GB-9 + YLL2 resulted in a significant increase in final weight, Docosahexaenoic acid (DHA) and Eicosapentenoic acid (EPA) concentration, compared with that of control (p < 0.05). For innate immunity, the results showed that skin mucus lysozyme activity, leukocytes phagocytosis activity and reactive oxygen species level, and serum alternative complement pathway activity, peroxidase and lysozyme activity were significantly higher in supplemented groups compared to the control (p < 0.05). The highest values were recorded in fish fed both YLL2 and GB-9 with respect to the individual application. The present results suggested that the combination of these supplementation could be considered as potential feed-additives for aquaculture farmed fish.