Relative Percent Survival Research Articles

Reverse vaccinology approach for the identifications of potential vaccine candidates against Salmonella

Salmonella is a leading cause of foodborne pathogen which causes intestinal and systemic diseases across the world. Vaccination is the most effective protection against Salmonella, but the identification and design of an effective broad-spectrum vaccine is still a great challenge, because of the multi-serotypes of Salmonella. Reverse vaccinology is a new tool to discovery and design vaccine antigens combining human immunology, structural biology and computational biology with microbial genomics. In this study, reverse vaccinology, an in-silico approach was established to screen appropriate immunogen targets by calculating the immunogenicity score of 583 non-redundant outer membrane and secreted proteins of Salmonella. Herein among 100 proteins identified with top-ranked scores, 15 representative antigens were selected randomly. Applying the sequence conservation test, four proteins (FliK, BcsZ, FhuA and FepA) remained as potential vaccine candidates for in vivo evaluation of immunogenicity and immunoprotection. All four candidates were capable to trigger the immune response and stimulate the production of antiserum in mice. Furthermore, top-ranked proteins including FliK and BcsZ provided wide antigenic coverage among the multi-serotype of Salmonella. The S. Typhimurium LT2 challenge model used in mice immunized with FliK and BcsZ showed a high relative percentage survival (RPS) of 52.74 % and 64.71 % respectively. In conclusion, this study constructed an in-silico pipeline able to successfully pre-screen the vaccine targets characterized by high immunogenicity and protective immunity. We show that reverse vaccinology allowed screening of appropriate broad-spectrum vaccines for Salmonella.

Roles of surface layer proteins in the regulation of Pediococcus pentosaceus on growth performance, intestinal microbiota, and resistance to Aeromonas hydrophila in the freshwater prawn Macrobrachium rosenbergii

Maintaining the balance of intestinal microbiota is vital for the growth and health of crustaceans. The current study explored the influences of the surface layer proteins (SLPs) of Pediococcus pentosaceus on the growth performance, disease resistance, and gut microbial community of the freshwater prawn Macrobrachium rosenbergii. After 28 days of feeding, obvious enhancements in the growth performance were found in the PF (a commercial basic diet + intact P. pentosaceus) group compared to the CF (a commercial basic diet, control) and RF (a commercial basic diet + LiCl-treated P. pentosaceus) groups. Meanwhile, the relative percent survival (RPS) of the PF group was found to be 82.1%, higher than the RF group (RPS = 63.2%) in the challenge test. There were noteworthy differences in the relative richness of Aeromonas among different treatments, and the PF group was the lowest. Whereas the relative abundance of Lactococcus between the CF and RF group did not markedly differences, which was obviously lower than the PF group. The prediction of bacterial phenotype indicated that the pathogenic potential of intestinal microbiota was decreased by increasing dietary SLPs-carrying P. pentosaceus. The results suggested that SLPs-carrying P. pentosaceus can improve growth, modulate intestinal microbiota, and increase the resistance of prawns against Aeromonas hydrophila infection.

Impacts of pineapple peel powder on growth performance, innate immunity, disease resistance, and relative immune gene expression of Nile tilapia, Oreochromis niloticus

An 8-week growth trial was conducted to examine the efficacy of pineapple peel powder (PAPP) on growth rate and immunity of Nile tilapia, O. niloticus. Three hundred Nile tilapia (20.91 ± 0.11 g) were fed five diets containing different levels of PAPP at 0, 10, 20, 30 and 40 g kg−1 PAPP, respectively. After four and eight weeks of the feeding trial, growth rates, and immune responses were tested. A challenge test using Streptococcus agalactiae and relative immune gene expression were performed after eight weeks of PAPP feeding. It was found that skin mucus and serum lysozyme, skin mucus and serum peroxidase, alternative complement, phagocytosis, and respiratory burst activities were significantly increased with the addition of PAPP. The maximum (P ≤ 0.05) innate immune values were noted in fish fed 10 g kg−1 PAPP. Similarly, the up-regulation of IL1, IL8, and LBP gene expressions were also detected in fish fed PAPP diets, with the maximum value was found in 10 g kg−1 PAPP fed fish. The relative percentage of survival (RPS) of Oreochromis niloticus after the challenge test were (56.00%, 72.00%, 60.00%, and 44.00%) for the 5, 10, 20 and 40 g kg−1 PAPP diets, respectively. Fish fed the 10 g kg−1 PAPP supplemented diet achieved the highest (P < 0.05) survival rate against S. agalactiae. Growth and feed efficiency were outstandingly (P < 0.05) enhanced in the PAPP groups. In conclusion, PAPP can be potentially used as a feed additive in Nile tilapia culture under Biofloc system.

Using red tilapia to control Cryptocaryon irritans infestations

Marine cultured fish in tropical and sub-tropical regions often suffer from white spot disease, caused by Cryptocaryon irritans infection. This can lead to mass mortality and economic loss. This study investigated the use of seawater-adapted red tilapias (Oreochromis niloticus × O. mossambicus) as scavengers for cleaning tomonts, to protect susceptible marine fish from C. irritans infection. The sensitivity of red tilapia to C. irritans infection, the effectiveness of red tilapia in consuming tomonts, and the efficacy for protecting the vulnerable Trachinotus ovatus from C. irritans infection were evaluated. The results showed that the susceptibility of red tilapia to C. irritans infection was significantly lower than T. ovatus and the rate of tomonts consumption by red tilapia was positively correlated with the number of tomonts in the seawater. The relative percent survival of T. ovatus infected with C. irritans and co-cultured with red tilapias (at a density of 19 red tilapias/m2) was 97.78% at the12 days post-infection. This study indicates that polyculture with red tilapia could be a potential alternative biocontrol strategy to eliminate tomonts, for controlling C. irritans reinfection.

Immuno-protective efficiency of feed-based whole-cell inactivated bivalent vaccine against Streptococcus and Aeromonas infections in red hybrid tilapia (Oreochromis niloticus × Oreochromis mossambicus)

Streptococcosis and motile aeromonad septicemia (MAS) are well-known diseases in tilapia culture, which cause mass mortality with significant economic losses. The development of feed-based bivalent vaccines in controlling these diseases has been initiated, however, the mechanisms of immunities and cross-protection in fish remain unclear. This study was conducted to assess the immuno-protective as well as the cross-protective efficacy of a newly developed feed-based bivalent vaccine against Streptococcus and Aeromonas infections in red hybrid tilapia. A total of five groups of fish were vaccinated orally through two different techniques; bivalent vaccine (inactivated Streptococcus iniae and Aeromonas hydrophila) sprayed on feed pellets (BS group); bivalent vaccine (inactivated S. iniae and A. hydrophila) incorporated in feed (BI group); monovalent inactivated S. iniae and A. hydrophila vaccine separately incorporated into feed as monovalent S. iniae (MS group) and monovalent A. hydrophila (MA group); and control group (without vaccine). The feed-based vaccine was delivered orally at 5% of body weight for five consecutive days. The booster doses were given in the same manner on weeks 2 and 6. Serum and skin mucus samples were collected to assess the IgM responses using indirect ELISA. The first administration of the feed-based vaccine stimulated the IgM levels that lasted until week 3, while the second booster ensured that the IgM levels remained high for a period of 16 weeks in the BI, MS and MA groups. The BI group developed a strong and significantly (P < 0.05) higher systemic and mucosal IgM responses against both S. iniae and A. hydrophila and also cross-protective antigen S. agalactiae and A. veronii compared to the BS and control groups. Quantitative real-time PCR results also showed that the relative expressions of IL-8, INF-γ and IgM in the BI immunized fish spleen, head kidney and hindgut exhibited various significant (P < 0.05) rising trends following both the vaccination and the challenge phase. On weeks 10, all fish were challenged through the intraperitoneal route, where relative percent survivals (RPS) of 82.22 ± 3.85% when challenged with S. iniae, 77.78 ± 3.85% when challenged with A. hydrophila and 77.78 ± 3.85% when co-challenged with both S. iniae and A. hydrophila were observed in the BI group, which were significantly higher (P < 0.05) compared to the other groups. The BI group also showed significantly (P < 0.05) higher partial cross-protections following challenges with S. agalactiae (RPS at 60.00 ± 6.67%) and A. veronii (RPS at 57.78 ± 7.70%). This study demonstrated that immunization with feed-based BI vaccine elicited immune responses that were capable of protecting red hybrid tilapia against streptococcosis and MAS.

Vaccine Efficacy of a Newly Developed Feed-Based Whole-Cell Polyvalent Vaccine against Vibriosis, Streptococcosis and Motile Aeromonad Septicemia in Asian Seabass, Lates calcarifer.

Multiple infections of several bacterial species are often observed under natural farm conditions. The infections would cause a much more significant loss compared to a single infectious agent. Vaccination is an essential strategy to prevent diseases in aquaculture, and oral vaccination has been proposed as a promising technique since it requires no handling of the fish and is easy to perform. This research attempts to develop and evaluate a potential feed-based polyvalent vaccine that can be used to treat multiple infections by Vibrios spp., Streptococcus agalactiae, and Aeromonas hydrophila, simultaneously. The oral polyvalent vaccine was prepared by mixing formalin-killed vaccine of V. harveyi, S. agalactiae, and A. hydrophila strains with commercial feed pellet, and palm oil as an adjuvant was added to improve their antigenicity. Thereafter, a vaccinated feed pellet was tested for feed quality analysis in terms of feed stability in water, proximate nutrient analysis, and palatability, safety, and growth performance using Asian seabass, Lates calcarifer as a fish host model. For immune response analysis, a total of 300 Asian seabass juveniles (15.8 ± 2.6 g) were divided into two groups in triplicate. Fish of group 1 were not vaccinated, while group 2 was vaccinated with the feed-based polyvalent vaccine. Vaccinations were carried out on days 0 and 14 with oral administration of the feed containing the bacterin at 5% body weight. Samples of serum for antibody and lysozyme study and the spleen and gut for gene expression analysis were collected at 7-day intervals for 6 weeks. Its efficacy in protecting fish was evaluated in aquarium challenge. Following vaccination by the polyvalent feed-based vaccine, IgM antibody levels showed a significant (p < 0.05) increase in serum against Vibrio harveyi, Aeromonas hydrophila, and Streptococcus agalactiae and reached the peak at week 3, 5, and 6, respectively. The high-stimulated antibody in the serum remained significantly higher than the control (p < 0.05) at the end of the 6 weeks vaccination trial. Not only that, but the serum lysozyme level was also increased significantly at week 4 (p < 0.05) as compared to the control treatment. The immune-related gene, dendritic cells, C3, Chemokine ligand 4 (CCL4), and major histocompatibility complex class I (MHC I) showed significantly higher expression (p < 0.05) after the fish were vaccinated with the oral vaccine. In the aquarium challenge, the vaccine provided a relative percentage survival of 75 ± 7.1%, 80 ± 0.0%, and 80 ± 0.0% after challenge with V. harveyi, A. hydrophila, and S. agalactiae, respectively. Combining our results demonstrate that the feed-based polyvalent vaccine could elicit significant innate and adaptive immunological responses, and this offers an opportunity for a comprehensive immunization against vibriosis, streptococcosis, and motile aeromonad septicemia in Asian seabass, Lates calcarifer. Nevertheless, this newly developed feed-based polyvalent vaccination can be a promising technique for effective and large-scale fish immunization in the aquaculture industry shortly.

Effects of dietary sweet basil (Ocimum basilicum) oil on the performance, antioxidants and immunity welfare, and resistance of Indian shrimp (Penaeus indicus) againstVibrio parahaemolyticusinfection

In the present study, Indian shrimp, Penaeus indicus, were fed on diets supplemented with basil (Ocimum basilicum) oil (BO) to observe the growth performance, antioxidant and innate immune responses, and the resistance against Vibrio parahaemolyticus infection. The animals (5.0–5.5 g) were fed on diets supplemented with 0.0 (control), 1.0, 2.5 and 5 g BO/kg diet over 90 days. After the feeding trial, animals were intramuscularly injected by V. parahaemolyticus and observed for 96 h for mortality or any abnormal signs. Growth-stimulating effects of dietary BO supplementation on final weight, weight gain percentage, specific growth rate and feed intake were observed. No significant differences were found in feed conversion ratio, protein efficiency ratio, protein productive value and animal survival. Feeding the animals on BO-enriched diets did not significantly affect the chemical proximate composition of animals’ muscles. Increased values of total protein, albumin and globulin were observed in BO-fed animals; meanwhile, aspartate and alanine aminotransferases as well as creatinine and urea levels significantly decreased in animals fed 2.5–5.0 g BO/kg diet. Activities of superoxide dismutase, catalase, glutathione peroxidase, lysozyme and phenol oxide activity significantly (p < .05) increased in the BO-fed fish, with highest values in treatments of 2.5–5.0 g/kg diet. The dietary BO supplementation significantly (p < .05) decreased levels of malondialdehyde and nitrous oxide. After the bacterial challenge, 66.7 percent of animals in the control group were dead. The BO-fed animals were more resistant against bacterial challenge especially at levels of 2.5–5.0 g/kg diet, which showed high relative percentage of survival (80.0–86.7 percent). In conclusion, dietary BO supplementation with levels of 2.5–5.0 g/kg diet proved beneficial effects to Indian shrimp where growth performance, antioxidant systems and innate immunity were stimulated. It also promoted the challenge of Indian shrimp against V. parahaemolyticus infection.

Effects of mixed probiotic (Lactobacillussp. andSaccharomyces cerevisiae) on the growth performance and immune gene expression of tilapia (Oreochromis niloticus) afterStreptococcus agalactiaevaccination

The farming of Nile tilapia (Oreochromis niloticus) has faced the challenges of infections, especially by Streptococcus However, solving this problem by using antibiotics will cause other problems, such as drug residue that can directly affect consumers and an increase in antibiotic resistance to bacteria. Therefore, probiotics should be an alternative to control bacterial diseases. This study aimed to investigate the optimum level of mixed probiotic supplementation. The experiment was designed to include six treatments (the control diet and probiotic-supplemented diets at 0.5, 1.0, 2.0, 5.0 and 10.0%). The mixed probiotic was composed of 3.1 × 108 CFU of Lactobacillus sp. and 4.6 × 106 CFU of Saccharomyces cerevisiae. After feeding for 30 days, tilapias from each replication were injected with a vaccine containing Streptococcus agalactiae to study the immune gene expression. The results showed that this mixed probiotic can enhance the growth and immune system of tilapias, and the most effective level was at 0.5% with consideration for high ADG, low FCR, and high protective efficiency against S. agalactiae infection. Moreover, toll-like receptor 9 gene and immunoglobulin gene expressions after vaccination for 24 hours were significantly higher than the control. The relative percent survival was the highest at 78.13%. This mixed probiotic can be used for Streptococcosis control in tilapia farms because it can reduce the diet cost and induce both the growth and immune system of tilapias.

Local immune responses to VAA DNA vaccine against Listonella anguillarum in flounder (Paralichthys olivaceus)

The efficacy of DNA vaccine is associated closely with the expression of the antigen and the intensity of local immune responses. In our previous study, a recombinant DNA plasmid expressing the VAA protein (pVAA) of Listonella anguillarum has been proved to have a good protection against the infection of L. anguillarum. In the present study, the local immune responses eliciting by immunizing flounder with intramuscular (I.M.) injection of pVAA was investigated at the cellular and genetic level, the muscle at the injection site at 7th post vaccination day was sampled and analyzed by hematoxylin-eosin (H&E) staining, immunohistochemistry (IHC), flow cytometry (FCM), RNA sequencing (RNA-Seq)-based transcriptomics and RT-qPCR. Then variations on the specific antibodies in serum at 1st–6th post vaccination week and the relative percent survival rate (RPS) at following 14 days after challenge were measured. The H&E results showed that inflammatory cells and immune cells significantly increased at the injection site. The IHC using monoclonal antibody against T cell markers revealed that both CD4-1+ and CD4-2+ T lymphocytes were recruited to the injection site and FCM results showed that the proportion of CD4-1+ cells in pVAA immunized group was 28.6 %, in the control group was 8.7 %, and that of CD4-2+ cells in two groups was 21.2 % and 8.5 %, respectively. These results indicating that the proportion of CD4+ cells in the immune group was significantly increased compared with the control group. Moreover, there were 2551 genes differently expressed in pVAA immunized group, KEGG analysis showed the genes involved in the signal transduction and immune system, and surface markers for B-cells genes, T-cells and antigen presenting cells (APCs) genes were highly upregulated, suggesting the activation of the systemic immune responses. Antibody specific anti-L. anguillarum or anti-rVAA antibodies were significantly induced at 2nd post-immunization week, that reached a peak at 4–5th week. RPS in pVAA group was 53.85±3.64 %. In conclusion, pVAA induced effective local immune responses and then the systematic response. This probably is the main contribution of pVAA to effective protection against L. anguillarum.

Modulation of the mucosal immune response of red tilapia (Oreochromis sp.) against columnaris disease using a biomimetic-mucoadhesive nanovaccine

Columnaris, a highly contagious bacterial disease caused by Flavobacterium columnare, is recognized as one of the most important infectious diseases in farmed tilapia, especially during the fry and fingerling stages of production. The disease is associated with characteristic lesions in the mucosa of affected fish, particularly their skin and gills. Vaccines delivered via the mucosa are therefore of great interest to scientists developing vaccines for this disease. In the present study, we characterized field isolates of F. columnare obtained from clinical columnaris outbreaks in red tilapia to select an isolate to use as a candidate for our vaccine study. This included characterizing its colony morphology, genotype and virulence status. The isolate was incorporated into a mucoadhesive polymer chitosan-complexed nanovaccine (CS-NE), the efficacy of which was determined by experimentally infecting red tilapia that had been vaccinated with the nanoparticles by immersion. The experimental infection was performed 30-days post-vaccination (dpv), which resulted in 89% of the unvaccinated control fish dying, while the relative percentage survival (RPS) of the CS-NE vaccinated group was 78%. Histology of the mucosal associated lymphoid tissue (MALT) showed a significantly higher presence of leucocytes and a greater antigen uptake by the mucosal epithelium in CS-NE vaccinated fish compared to control fish and whole cell vaccinated fish, respectively, and there was statistically significant up-regulation of IgT, IgM, TNF α, IL1-β and MHC-1 genes in the gill of the CS-NE vaccinated group. Overall, the results of our study confirmed that the CS-NE particles achieved better adsorption onto the mucosal surfaces of the fish, elicited great vaccine efficacy and modulated the MALT immune response better than the conventional whole cell-killed vaccine, demonstrating the feasibility of the mucoadhesive nano-immersion vaccine as an effective delivery system for the induction of a mucosal immune response against columnaris disease in tilapia.

Ozone nanobubble modulates the innate defense system of Nile tilapia (Oreochromis niloticus) against Streptococcus agalactiae

Ozone nanobubble (NB–O3) is a promising technology for improving dissolved oxygen and reducing bacterial concentration in aquaculture systems. Here, we investigated the effects of NB-O3 on the innate immunity of fish by monitoring the expression levels of nonspecific immune-related genes (IL-1β, IL-2β, TNF-α), heat-shock protein genes (HSP70, HSP90-α), and a bacteriolytic enzyme, C-type lysozyme, gene (LYZ) post-treatment with this technology. Following exposure to NB-O3, the different tissues of Nile tilapia (Oreochromis niloticus) were collected over time for quantitative real-time PCR (qPCR) analysis. The expression of all the genes evaluated in the gills, the head kidney, and the spleen of the NB-O3 treated group was significantly up-regulated compared to that in the untreated control group. The expression levels were the highest (approx. 2 to 4-fold) at 15 min and 3 h post-exposure and then decreased from 6 to 24 h. These findings suggested that NB-O3 could switch on the innate immunity genes of Nile tilapia. Thus, we hypothesized that the NB-O3-immune-activated fish would respond more effectively to subsequent bacterial infections, thereby improving survivability compared to that of untreated fish. To test this hypothesis, 3 h post NB-O3 exposed fish and unexposed fish were challenged with a lethal dose of Streptococcus agalactiae. Interestingly, the survival rate of the NB-O3 group was significantly higher than that of the non-treated controls, with a relative percent survival (RPS) of 60–70%. Together, these findings indicate, for the first time, that NB-O3 may trigger the nonspecific defense system of the fish, thereby improving fish survivability during subsequent bacterial infections. This research identified another potential benefit of NB-O3 in aquaculture for preventing infectious bacterial diseases.

The effect of natural and artificial periphytic substrates with biofloc system on shrimp Penaeus vannamei (Boone 1931) culture: growth and immune response

A 120-day experiment was conducted to understand the growth and immunomodulatory response of Penaeus vannamei reared in biofloc-integrated periphyton system–like natural substrates like bamboo (NS1), sugarcane bagasse (NS2), artificial substrates like polyvinyl chloride mat (AS1), agricultural shed net (AS2), and a control group without biofloc and substrate. Experiments were carried out in 500-L fiber-reinforced plastic (FRP) tanks at a stocking density of 100 nos/m3 (0.256 g). Shrimps reared in natural substrate-based treatments, (NS1 and NS2) recorded highest average body weight (ABW) and survival followed by artificial ones (AS1 and AS2), and were significantly higher (P < 0.01) compared with control. Significantly higher chlorophylla, ash-free dry matter (AFDM), and THB load were recorded in NS (1 and 2) followed by AS (1 and 2). Contrastingly, the Vibrio load was higher in control than treatments. Relative percentage survival was higher (P < 0.01) in treatments when challenged against V. parahaemolyticus. Serum protein level, phenoloxidase activity, and superoxide dismutase assay were significantly higher in natural and artificial substrate groups when compared to control. Immunomodulatory effects through immune gene study revealed a two fold to four fold upregulation in cytMn-SOD and mtMn-SOD genes in NS groups. Similarly, the lysozyme and prophenoloxidase genes were significantly upregulated in the treatment groups. The results conclude that biofloc with natural followed by artificial periphytic substrates were observed to deliver better performances, protective response, and immunomodulatory effects in pacific white shrimp P. vannamei culture.

Antioxidant defence system based oxidative stress mitigation through dietary jamun tree leaf in experimentally infected snubnose pompano, Trachinotus blochii.

A 45-day feeding trial was conducted to evaluate the effect of dietary jamun tree leaf (JL) on the antioxidant defence system-based disease resistance in juveniles of Trachinotus blochii. The juveniles of snubnose pompano were distributed into four treatment groups in triplicates. Each treatment was fed with a diet containing either 0 (0JL), 0.5 (0.5JL), 1 (1JL) and 1.5% JL (1.5JL) in the feed. After feeding trial, the fishes were experimentally infected with Vibrio parahaemolyticus. The activities of oxidative stress enzymes such as superoxide dismutase and catalase were found to be increasing with increasing level of dietary JL incorporation, and the lower value was witnessed in control group in pre- and post-challenge. After challenge, the alanine and aspartate aminotransferase activities in all the treatments were significantly increased (P < 0.05) than the pre-challenge condition and exhibited reverse trend with the antioxidant enzymes. The alkaline and acid phosphatase activities were found higher in 1.5JL group and showed significant difference (P < 0.05) among the treatments. The respiratory burst activity and liver glycogen content showed an increasing trend as the level of inclusion of JL increased in the diet. The acetylcholinesterase activity was significantly plunged (P < 0.05) after experimental infection, and JL diet fed groups showed better activity. After experimental infection with V. parahaemolyticus, the highest relative percentage of survival was observed in 1JL and 1.5JL groups. Hence, dietary supplementation of jamun tree leaf at the level of 1% is adequate to reduce the oxidative stress and improved the innate immune status through antioxidant defence system.

Isolation, identification and vaccine development of serotype III Streptococcus parauberis in turbot (Scophthalmus maximus) in China

Streptococcus parauberis is an important disease in farming fish that could infect a series of fish causing streptococcosis, which leads to significant economic losses. In this research, we first characterized the streptococcosis caused by S. parauberis in farming fish in China. In 2019, bacteria were isolated from diseased turbot in 3 farms located in Penglai, Haiyang, and Laizhou city, Shandong Province, and identified as S. parauberis by 16S rRNA sequencing and Biolog GenIII. Capsule polysaccharide (cps) genes of isolates were obtained by whole genome sequencing and aligned with the cps cluster in GenBank. By cps genes analysis, the isolates were all classified as serotype III, which were different from the isolates that infect flounder (Paralichthys olivaceus) in Japan and South Korea, but the same with the isolates in Spain and the USA reported recently. Then the serotype III S. parauberis vaccines were developed for turbot. The specific antibody could be detected in the vaccinated fish, and the specific antibody from oil emulsified vaccine group fish was significantly higher than that of the water-based vaccine group from 14 days post vaccination. The relative percent of survival were 78.00% and 100% for water-based vaccine and Marcol 52 emulsified vaccine, respectively. To our knowledge, this is the first report of S. parauberis infection in farming fish in China, and would provide important information for disease control and prevention for streptococcosis in aquaculture.

Antioxidant and antibacterial activity of Gelidium pusillum (Stackhouse) against Aeromonas caviae and its applications in aquaculture

Over the past two decades, intensive work has been conducted on antioxidants as they have great potential to prevent diseases and complications associated with age. Seaweeds are rich natural sources of bioactive metabolites with antioxidant and antimicrobial characteristics. In the present study, bioactive metabolites were extracted from the seaweed Gelidium pusillum by maceration in aqueous and ethanolic medium. The dried weed powder was analyzed by GC-MS and FTIR. The macerated products were assayed for antioxidant and antibacterial activity by in vitro and in vivo methods. Antibacterial activity against the marine pathogen Aeromonas caviae was observed. Giant freshwater prawns (Macrobrachium rosenbergii) were used as models for in vivo assays by inducing pathogenicity by intramuscular injection of pathogen. The present study revealed that G. pusillum is a rich source of antioxidant and antibacterial compounds. The data obtained from the present study substantiates the potential of G. pusillum as an ideal antioxidant and antibacterial in the aquaculture industry. The animals treated with the ethanolic extract of G. pusillum (35 μl of 1mg/ml extract) survived even after 3 weeks, compared to the untreated in which 100% mortality was observed within 120 h. Relative percentage survival (RPS) of 90% was observed in the group treated with the seaweed extract. GC-MS analysis revealed the presence of several compounds previously reported for antibacterial activity. In the present work, a total of seven compounds were identified by GC-MS analysis, out of which two compounds are reported to have antimicrobial activity. High sustained survival of the infected prawns post-administration of the extract revealed that this sea weed has potential in the biopharmaceutical, aquaculture, and food sectors.

Study on Immune Response of Organs of Epinephelus coioides and Carassius auratus After Immersion Vaccination With Inactivated Vibrio harveyi Vaccine.

Immersion vaccination relies on the response of fish mucosa-associated lymphoid tissues, the Crucian carp (Carassius auratus) and Grouper (Epinephelus coioides) were researched in this paper to examine local mucosal immune responses and associated humoral system responses following immersion vaccination. We administered 1.5 × 107 CFU/ml formalin-inactivated Vibrio harveyi cells and measured mucus and serum antibody titers as well as IgM, MHC II mRNA levels in immune organs. The mucosal antibody response preceded the serum response indicating a role for local mucosal immunity in immersion vaccination. IgM and MHC II mRNA levels were relatively greater for the spleen and head kidney indicating the importance and central position of systemic immunity. Expression levels were also high for the gills while skin levels were the lowest. IgM and MHC II mRNA levels were altered over time following vaccination and the hindgut, liver and spleen were similar indicating a close relationship, so the absolute value of r is used to analyze the correlation among different organs immunized. It can be inferred the existence of an internal immune molecular mechanism for Immune synergy hindgut-liver-spleen, from the peak time (14th day), the relative ratio of genes expression in the same tissues between the immunized grouper and the control group (26 times), and Pearson correlation coefficient (0.8<|r|<1). Injection challenges with live V. harveyi indicated that the relative protection rates for the crucian carp and Grouper was basically the same at 44.4% and 47.4%, respectively. It is believe that crucian carp may be used as a substitute for the valuable grouper in immunity experiment, just from aspect of the relative percent survival (RPS) and how it changes with time. But they were not consistent about the IgM mRNA expression between that of crucian carp and grouper after immersion the Vibrio vaccine.

Effects of a short pulse administration ofUlva rigidaon innate immune response and intestinal microbiota inSparus auratajuveniles

Given the potential of algae as a new aquafeed ingredient, this study evaluated the effect of the tank-cultivated macroalgae Ulva rigida C. Agardh (Chlorophyta) on innate immune response, intestinal microbiota and their resistance against Photobacterium damselae subsp. piscicida in juvenile gilthead seabream (Sparus aurata L.) after a short pulse of administration. An algae-free diet was used as control, and an experimental diet was formulated containing 25% of U. rigida (UL-25). After 2, 7, 15 and 30 days biological samples were taken. No statistical significance (p > 0.05) was detected in respiratory burst activity. Diet UL-25 significantly enhanced alternative complement pathway activity respect to control diet from the beginning of the experiment (p < 0.05). When fish were challenged against P. damselae subsp. piscicida, UL-25-fed fish were more resistant against the pathogen than the fish fed control diet, obtaining a relative survival percentage of 54.55%. The ability of UL-25 diet to modify the intestinal microbiota was also confirmed. In conclusion, the use of a short pulse of dietary administration of 25% U. rigida in S. aurata diet modulates the immune response, intestinal microbiota and reduced the mortality rate during P. damselae subsp. piscicida infection.

Boswellia serrata Resin Extract in Diets of Nile Tilapia, Oreochromis niloticus: Effects on the Growth, Health, Immune Response, and Disease Resistance to Staphylococcus aureus.

Simple SummaryThe current study evaluated the effects of Boswellia serrata resin extract (BSRE) as a feed additive on the growth performance, immune response, antioxidant status, and disease resistance of Nile tilapia, Oreochromis niloticus. Fish were fed on four basal diets complemented with four levels of BSRE 0, 5, 10, or 15 g kg−1. The results of this study proposed that BSRE addition can enhance the antioxidant activity, immune status, and disease resistance of O. niloticus to S. aureus infection. The level of 5 g kg−1 BSRE can improve fish growth without causing harmful effects on fish health. Higher levels of BSRE are not recommended as they badly affected the histoarchitecture of many vital organs.The influences of Boswellia serrata resin extract (BSRE) as a feed additive on the growth performance, immune response, antioxidant status, and disease resistance of Nile tilapia, Oreochromis niloticus L. were assessed. One hundred-forty four fingerlings (initial weight: 21.82 ± 0.48 g) were randomly allotted into four groups with three replicates where they were fed on one of four treatments with four levels of Boswellia serrata resin extract 0, 5, 10, or 15 g kg−1, BSRE0, BSRE5, BSRE10, BSRE15, respectively for eight weeks. After the end of the feeding trial, the fish were challenged with Staphylococcus aureus, and mortalities were noted. The final body weight, total body weight gain, and the total feed intake were quadratically increased in BSRE5 treatment (p < 0.01). The protein productive efficiency (PPE) was linearly and quadratically increased in all BSRE supplemented treatments (p < 0.01). Dietary addition of BSRE raised the fish crude protein content and reduced the fat content in a level-dependent manner (p < 0.01). The ash content was raised in the BSRE15 group (p < 0.01). Dietary BSRE supplementation decreased the serum levels of glucose, total cholesterol, triglycerides, and nitric oxide. It increased the serum levels of total protein, albumin, total globulins, α1 globulin, α2 globulin, ß globulin, ɣ globulin, Catalase, and SOD (superoxide dismutase) activity, GSH (reduced glutathione), lysozyme activity, and MPO (myeloperoxidase) in a level-dependent manner (p < 0.05). The BSRE15 diet increased the serum level of ALT (alanine aminotransferase) and decreased creatinine serum level (p < 0.05). Dietary BSRE supplementation increased the relative percentage of survival % (RPS) of S. aureus challenged fish. The histoarchitecture of the gills and kidney was normal in the BSRE5 treatment and moderately changed in BSRE10 and BSRE15 treatments. The splenic lymphoid elements were more prevalent, and the melano-macrophage centers (MMC) were mild to somewhat activated in BSRE supplemented treatments. Dietary BSRE supplementation improved the intestinal histomorphology. It can be concluded that BSRE addition can enhance the antioxidant activity, immune status, and disease resistance of O. niloticus to S. aureus infection. The level of 5 g kg−1 BSRE can improve fish growth without causing harmful effects on fish health. The highest levels of BSRE are not recommended as they badly affected the histoarchitecture of many vital organs.

Protective efficacy of four heat-shock proteins as recombinant vaccines against photobacteriosis in Asian seabass (Lates calcarifer)

Photobacterium damselae subsp. piscicida (Phdp) is the causative agent of photobacteriosis in marine fish and is responsible for huge losses to marine aquaculture worldwide. Efforts have been made to develop a vaccine against this disease. Heat-shock proteins (HSPs) are a family of proteins that are ubiquitous in cellular life. Bacteria produce elevated levels of HSPs as a survival strategy when exposed to stressful environments in a host during infection. This group of proteins are also important antigens that can induce both humoral and cellular immune responses. In this study, four HSPs of Phdp, HSP90, HSP33, HSP70, and DnaJ, were selected for cloning and recombinant expression. Western blotting with rabbit anti-Phdp helped identify rHSP70 and rHSP33 as immunogenic proteins. Asian seabass (Lates calcarifer) immunised with rHSP90, rHSP33, rHSP70, and rDnaJ showed 48.28%, 62.07%, 51.72%, and 31.03% relative percent survival, respectively, after being challenged with Phdp strain AOD105021. High expression levels of immune-related genes and high antibody titres were observed in the rHSP33 group, and the sera of this group also exhibited a high level of bactericidal activity against Phdp. Collectively, our results suggest that HSP33 is a potential candidate for vaccine development against Phdp infection.

Recombinant resuscitation-promoting factor protein of Nocardia seriolae, a promissing vaccine candidate for largemouth bass (Micropterus salmoides)

Nocardia seriolae is an important pathogenic bacterium that causes nocardiosis in various fish species and leads to economic losses in the fish industry. To develop an effective subunit vaccine against nocardial infection, the truncated resuscitation-promoting factor (tRPF) of N. seriolae was selected and recombinantly produced using the Escherichia coli expression system. Western blotting results indicated that the recombinant protein could be strongly recognised by largemouth bass anti-N. seriolae antibodies. The protective efficacy of tRPF recombinant protein was assessed in combination with the commercial adjuvant Montanide™ ISA 763 A VG. The results showed that emulsified tRPF + ISA significantly induced high serum antibody response and serum lysozyme activity in the vaccinated fish. Quantitative reverse transcription polymerase chain reaction analysis indicated that tRPF + ISA could notably enhance the expression of immune-related genes in both the head kidney and spleen of the vaccinated fish. Finally, vaccinated largemouth bass displayed higher immuno-protection with a relative percent survival of 69.23% compared to the control groups. Taken together, the combination of tRPF + ISA is an ideal vaccine candidate against N. seriolae infection.