Live recombinant Lactococcus lactis vaccine expressing aerolysin genes D1 and D4 for protection against Aeromonas hydrophila in tilapia (Oreochromis niloticus)

Aims: To evaluate a live recombinant Lactococcus lactis vaccine expressing aerolysin genes D1 (Lac‐D1ae) and/or D4 (Lac‐D4ae) in protection against Aeromonas hydrophila in tilapia (Oreochromis niloticus). Methods and Results: The polymerase chain reaction (PCR)‐amplified 250‐ and 750‐bp sequences coding for domains D1 and D4 of aerolysin were individually cloned into pNZ8048 and electrotransformed into L. lactis. The recombinant vaccine candidates were then either orally fed or injected intraperitoneally into tilapia. The development of antibodies in sampled fish compared to control groups implied that the recombinant epitopes expressed in L. lactis were able to elicit an immunogenic response in tilapia. Interestingly, the lower doses of both Lac‐D1ae and Lac‐D4ae gave higher antibody levels over the study period. Fish immunized with Lac‐D1ae and Lac‐D4ae together showed the highest level of protection, and the mortality was reduced significantly compared to control strains in both modes of vaccination. Conclusions: The recombinant L. lactis strain expressing D1 and D4 produced aerolysin‐specific serum IgM in tilapia. Both D1 and D4 promoted 55–82% relative per cent survival (RPS) against Aeromonas infection through intraperitoneal injection, whereas the RPS following oral feeding of the vaccine was 70–100%. Significance and Impact of the Study: The D1 and D4 regions of the aerolysin protein have been successfully identified as immunogenic regions that can elicit antibody production in tilapia and protect against challenge with Aer. hydrophila. A promising oral vaccine using L. lactis harbouring the D1 and D4 regions has been developed to control Aer. hydrophila.     

A 14 000-year record of paleoenvironmental change in the western basin of China's third largest lake,Lake Taihu

Effect of leaf extract of Ocimum sanctum on (i) the specific and non-specific immune responses and (ii) disease resistance against Aeromonas hydrophila was investigated in Oreochromis mossambicus. Sheep red blood cells (SRBC) and Heat aggregated bovine serum albumin (HA-BSA) were used as antigens for specific and non-specific immune response studies, respectively. Antigens were administered through intraperitoneal route. Anti-SRBC antibody titres were determined by direct haemagglutination and anti-bacterial antibodies were determined by bacterial agglutination. Nitroblue tetrazolium (NBT) assay was used to determine neutrophil activity. Disease resistance was measured in terms of relative percent survival (RPS). The immunostimulatory effect of the leaf extract of O. sanctum, when administered through intraperitoneal and oral routes was obvious. Leaf extract of O. sanctum, when administered intraperitoneally, stimulated both antibody response and neutrophil activity. Dietary intake of O. sanctum also enhanced the antibody response and disease resistance against A. hydrophila. Possibility of using O. sanctum as immunostimulant in the maintenance of finfish health in intensive freshwater aquaculture is suggested.     

Protective immunity by oral immunization with heat‐killed Shigella strains in a guinea pig colitis model

The protective efficacy of and immune response to heat‐killed cells of monovalent and hexavalent mixtures of six serogroups/serotypes of Shigella strains (Shigella dysenteriae 1, Shigella flexneri 2a, S. flexneri 3a, S. flexneri 6, Shigella boydii 4, and Shigella sonnei) were examined in a guinea pig colitis model. A monovalent or hexavalent mixture containing 1 × 10⁷ of each serogroup/serotype of heat‐killed Shigella cells was administered orally on Days 0, 7, 14 and 21. On Day 28, the immunized animals were challenged rectally with 1 × 10⁹ live virulent cells of each of the six Shigella serogroups/serotypes. In all immunized groups, significant levels of protection were observed after these challenges. The serum titers of IgG and IgA against the lipopolysaccharide of each of the six Shigella serogroups/serotypes increased exponential during the course of immunization. High IgA titers against the lipopolysaccharide of each of the six Shigella serogroups/serotypes were also observed in intestinal lavage fluid from all immunized animals. These data indicate that a hexavalent mixture of heat‐killed cells of the six Shigella serogroups/serotypes studied would be a possible broad‐spectrum candidate vaccine against shigellosis.     

Coriolus versicolor polysaccharides enhance the immune response of crucian carp (Corassius auratus gibelio) and protect against Aeromonas hydrophila

To investigate the effect of Coriolus versicolor polysaccharides (CVPS) on the immune response of crucian carp (Corassius auratus gibelio), fish were fed diets containing CVPS at different concentrations including 0.5, 1.0 and 2.0 g kg^?1 for 5 weeks. Other groups of fish were vaccinated by intraperitoneal injection (i.p.) against Aeromonas hydrophila with a killed bacterin at the beginning of the experiment and fed the same diets as described above. Additionally, control fish and vaccinated‐only fish were fed basal diets without CVPS supplementation. The phagocytosis, lysozyme activity, complement components C3 and C4, E‐C3bRR (Erythrocyte‐C3b rosette rate) and E‐CIRR (Erythrocyte‐immune complex rosette rate) levels and circulating antibody titers in the serum were monitored. Five weeks after feeding the prescribed diet, fish were challenged with A. hydrophila and the mortalities recorded. Results showed that feeding non‐vaccinated and vaccinated crucian carp with CVPS stimulated the phagocytosis of leukocytes, lysozyme, complement components C3 and C4, erythrocyte immune adherence, and circulatory antibody titers in serum in vaccinated crucian carp. Immune parameters increased to their highest levels after 3 weeks of feeding the diet containing 0.5 or 1.0 g kg^?1 CVPS. These doses also resulted in the highest protection in the challenge experiment. Best survival (85%) was in the vaccinated group fed the diet containing 1.0 g kg^?1 CVPS, whereas almost 80% of control fish (negative control) and 50% of vaccinated‐only fish (positive control) died.     

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.     

High yield expression of an AHL-lactonase from <Emphasis Type="Italic">Bacillus</Emphasis> sp. B546 in <Emphasis Type="Italic">Pichia pastoris</Emphasis> and its application to reduce <Emphasis Type="Italic">Aeromonas hydrophila</Emphasis> mortality in aquaculture

Background  

Aeromonas hydrophila is a serious pathogen and can cause hemorrhagic septicemia in fish. To control this disease, antibiotics and chemicals are widely used which can consequently result in "superbugs" and chemical accumulation in the food chain. Though vaccine against A. hydrophila is available, its use is limited due to multiple serotypes of this pathogen and problems of safety and efficacy. Another problem with vaccination is the ability to apply it to small fish especially in high numbers. In this study, we tried a new way to attenuate the A. hydrophila infection by using a quorum quenching strategy with a recombinant AHL-lactonase expressed in Pichia pastoris.     

Characterization of Aeromonas hydrophila Strains of Clinical,Animal, and Environmental Origin Expressing the O:34 Antigen

A collection of Aeromonas strains of different origins were characterized for isolates expressing the O:34 somatic antigen. Of over 200 strains tested, approximately 14% belonged to serogroup O:34 with >85% of these strains identified as A. hydrophila regardless of source. A subset of 14 A. hydrophila O:34 strains were further analyzed for a number of structural and pathogenic features. Most O:34 strains expressed similar whole-cell protein profiles with regards to minor bands, but major band differences were noted in outer membrane proteins (OMPs) migrating between the 31K and 58K region. OMP profiles could be subdivided into three distinct patterns. All O:34 strains expressed a heterogeneous O polysaccharide side chain profile in their lipopolysaccharide (LPS), although some variation in the electrophoretic migration of lower and higher molecular weight LPS bands was noted. Polyclonal antisera raised against a 45-K OMP-associated protein of one O:34 strain (AH-195) reacted in immunoblot assays with a major 43 to 46-K OMP in 11 of 14 (79%) O:34 strains tested. Most O:34 strains (69%) were found to be pathogenic in mice with LD-50 values (i.p.) of <1.0 × 10⁷ CFU; pathogenicity appeared to correlate best with elevated protease activity. The collective results suggest significant differences in both structural and pathogenic properties between some members of the O:34 group originating from human and nonhuman (fish, water) sources. Received: 14 December 1995 / Accepted: 22 January 1996     

Isolation and characterization of new potential probiotic bacteria based on quorum-sensing system

Aims: This work was aimed at identifying strains which can degrade quorum‐sensing (QS) molecules from fish gut, with properties suitable for use as probiotic in aquaculture. Methods and Results: A total of 200 strains were obtained from the intestine gut of Carassius auratus gibelio after enrichment in KG medium contained 500 μg l^?1 of C6‐HSL as the sole source of carbon and nitrogen; one strain named QS inhibitor (QSI)‐1 was identified as the genus Bacillus spp. by morphological phenotypes, and the strain also possessed an aiiA homologue gene using PCR amplification. In vitro, QSI‐1 strongly interfered with violacein production by Chromobacterium violaceum. Coculture of QSI‐1 with fish pathogen effectively reduced the amount of acyl‐homoserine lactones (AHLs) and the extracellular proteases activity of Aeromonas hydrophila YJ‐1. The oral LD50 of QSI‐1 to fish was more than 10¹¹ CFU shown that it was avirulent to fish. Fish fed diet supplemented with QSI‐1 had good survival, suggesting that QSI‐1 showed protection against Aer. hydrophila infection. Conclusions: The results indicate that the isolate QSI‐1 might have the potential possibility to be used as a probiotic in aquaculture. Significance and Impact of the Study: This is the first report to describe a bacterium isolated from the intestine gut of C. auratus gibelio which can degrade AHLs and has the probiotic characteristics for its use in aquaculture.     

Immunostimulating effect of triiodothyronine: dietary administration of triiodothyronine in rohu (Labeo rohita) enhances immunity and resistance to Aeromonas hydrophila infection

The immunomodulatory effects of dietary administration of 3,3^′,5‐triiodo‐l ‐thyronine (T₃) at 0, 1, 5 and 10 mg  kg^?1 of diet for 60 days in rohu (Labeo rohita) fingerlings were studied. Oral administration of T₃ at all dose levels resulted in significantly (P<0.05) higher serum T₃ levels, total serum protein and globulin levels, and reduced albumin–globulin ratio (A : G) compared with the control group, whereas feeding of T₃ at 5 and 10 mg kg^?1 diet enhanced the growth and superoxide production by neutrophils. At the end of the 60‐day experimental period the optimum dosage of T₃ appeared to be the 5 mg  kg^?1 diet for rohu fingerlings, resulting in a significantly higher specific antibody titre against formalin‐killed Aeromonas hydrophila and lowering the mortality percentage against the A. hydrophila challenge.     

Antagonism of Aeromonas hydrophila by propolis and its effect on the performance of Nile tilapia, Oreochromis niloticus

Propolis, a resinous substance collected by Apis mellifera bees from various plant sources and mixed with secreted beeswax, is a multifunctional material used by bees in the construction, maintenance, and protection of their hives. The collected propolis sample, from High Egypt, was dark-green with olive-odor. The minimal inhibition concentration (MIC) of propolis-ethanolic-extract, against Aeromonas hydrophila, was 80 μg Propolis-ethanolic-extract and crude propolis (1%) were added to artificial basal diet with (30% crude protein) to evaluate their efficacy on the fish growth-performance, immunostimulation and resistance to A. hydrophila. Two hundred and twenty-five Oreochromis niloticus (8 ± 0.45 g/fish) were divided into three equal treatments (T) of triplet replicates. The fish of T₁ were fed on basal diet (control). The fish of T₂ were given the basal diet, containing propolis-ethanolic-extract. The fish of T₃ were given the basal diet containing crude propolis for 28 day. The fish were intraperitoneally challenged by A. hydrophila (0.2 × 10⁷ cells ml⁻¹) at the end of the feeding period and kept for 15 more days.The best growth rate and feed conversion ratio were obtained with T_2. The increase in the average daily gain, specific growth rate and feed efficiency ratio were highly significances in T₂ followed by T₃ when compared with the control group. The HCT-level and monocyte-counts were increased (T₂). No significant change, in the large lymphocytic-count was found among the three treatments (28–27–28%), while the neutrophil-count was significantly decreased (7%) with T₂ and increased (13.11%) with the control. A significant increase in serum lysozyme and serum bactericidal activities was found with T₂. The RLP against A. hydrophila was high with T₂ and T₃.The propolis-ethanolic-extract enhanced the growth, immunity and resistance of O. niloticus against A. hydrophila more than the crude propolis.     

Application of attenuated Lactococcus garvieae strain lacking a virulence-associated capsule on its cell surface as a live vaccine in yellowtail Seriola quinqueradiata Temminck and Schlegel

An attenuated Lactococcus garvieae strain lacking a virulence‐associated capsule on its cell surface was evaluated for application as a live vaccine. The attenuated strain (MS93003A) was obtained from the parent strain (MS93003V), which produced a well‐developed capsule, by culturing on an agar medium supplemented with 2,3,5‐triphenyltetrazolium chloride. When live cells of L. garvieae (MS93003A) or formalin‐killed cells (MS93003A) were used as an injectable vaccine, protection against virulent L. garvieae (MS93003V) was conferred on Seriola quinqueradiata. The MS93003A cells did not recover their virulence even after in vivo passages in fish. MS93003A live cells also conferred long‐lasting protective immunity to S. quinqueradiata against virulent L. garvieae infection.     

Immunization with heat‐inactivated Staphylococcus aureus induced an antibody response mediated by IgG1 and IgG2 in patients with recurrent tonsillitis

Currently Staphylococcus aureus is the predominant pathogen isolated from the respiratory tract of patients with recurrent tonsillitis. Because of an increase in multi‐drug resistant strains of S. aureus, there is a pressing need for effective treatments and preventive approaches to reduce the risk of invasive and life‐threatening infections. A preventive vaccine against S. aureus would have a tremendous clinical impact. However, multiple clinical trials have failed to identify an agent that can induce protective responses. Most trials have been based on subunit vaccines using one or a few purified antigens, which may not be enough to confer protection. Here, the impact of a whole‐cell vaccine comprised of heat‐inactivated S. aureus was investigated in patients with RT. The vaccine was well tolerated and had no significant local or systemic reactions. Immunization with heat‐inactivated S. aureus elicited a significant antibody response characterized by production of IgG1 and IgG2 antibodies and, to a lesser extent, of IgA antibodies. Notably, this response was associated with an important decrease in the incidence of tonsillitis and bacterial colonization of the oropharyngeal mucosa. Our results show that whole‐cell inactivated S. aureus is safe and capable of evoking specific antibody responses in patients with recurrent tonsillitis.     

The role of flagella and motility in the adherence and invasion to fish cell lines by Aeromonas hydrophila serogroup O:34 strains

We compared the ability of Aeromonas hydrophila wild-type strains of serogroup O:34, non-motile Tn5 aflagellar mutants and the same mutants harboring a recombinant cosmid DNA from a library of A. hydrophila AH-3 (O:34, wild-type) that allows these mutants to make flagella and to be motile, to adhere and invade two fish cell lines. We found that motility is essential in these strains for adhesion, and also that possession of flagella is essential for the ability to invade the fish cell lines. We cannot rule out that flagella may be an adhesin, or that motility may also be involved in A. hydrophila serogroup O:34 bacterial invasion of both fish cell lines.     

Detection of aerolysin gene in <Emphasis Type="Italic">Aeromonas hydrophila</Emphasis> isolated from fish and pond water

Aerolysin is a hemolytic toxin encoded by aerolysin gene (1482 bp) that plays a key role in the pathogenesis of Aeromonas hydrophila infection in fish. New speciesspecific primers were designed to amplify 326 bp conserved region of aerolysin gene for A. hydrophila. Twenty-five isolates of A. hydrophila recovered from fish and pond water were studied for detection of aerolysin gene. Aerolysin gene was detected in 85% of the isolates during the study. The designed primers were highly specific and showed no cross reactivity with Escherichia coli, Aeromonas veronii, Vibrio cholerae, Flavobacterium spp., Chyseobacterium spp. and Staphylococcus aureus. The sensitivity limit of primers for detection of aerolysin gene in the genomic DNA of A. hydrophila was 5 pg.     

Protein fingerprinting profiles in different strains of <Emphasis Type="Italic">Aeromonas hydrophila</Emphasis> isolated from diseased freshwater fish

Summary Aeromonas hydrophila(Ah) strains isolated from diseased fish in India were studied for protein profiling using the SDS-PAGE protein fingerprinting profile pattern of whole cells of 12 local strains of A. hydrophilaand one reference strain (MTCC 646). Variability among the strains was observed. The average similarity between the 12 strains of A. hydrophila ranged from 0.272 to 0.916. Proteins with molecular mass of 55.6 and 14.67 kDa in Ah1, Ah2 and Ah3, 28.5 and 27.9 kDa in Ah4, Ah5 and Ah6, 21.4 and 19.5 kDa in Ah7, Ah8, Ah9 and 72.9, 91.5 and 71.3 kDa in Ah10, Ah11 and Ah12 were common. The protein polypeptide bands from 19.5 to 86.2 kDa were common in both local strains and reference strain of A. hydrophila. The protein fingerprinting study showed that there is genetic similarity between strains of A. hydrophila and reference strain (MTCC 646). These protein markers may be useful for further strain differentiation in epidemiological study.     

In vitro and in vivo efficacy of rosmarinic acid on quorum sensing mediated biofilm formation and virulence factor production in Aeromonas hydrophila

Rosmarinic acid (RA) was assessed for its quorum sensing inhibitory (QSI) potential against Aeromonas hydrophila strains AH 1, AH 12 and MTCC 1739. The pathogenic strains of A. hydrophila were isolated from infected zebrafish and identified through biochemical analysis and amplification of a species-specific gene (rpsL). The biofilm inhibitory concentration (BIC) of RA against A. hydrophila strains was found to be 750 μg ml^?1. At this concentration, RA reduced the QS mediated hemolysin, lipase and elastase production in A. hydrophila. In FT-IR analysis, RA treated A. hydrophila cells showed a reduction in cellular components. Gene expression analysis confirmed the down-regulation of virulence genes such as ahh1, aerA, lip and ahyB. A. hydrophila infected zebrafish upon treatment with RA showed increased survival rates. Thus, the present study demonstrates the use of RA as a plausible phytotherapeutic compound to control QS mediated biofilm formation and virulence factor production in A. hydrophila.     

Effect of dietary β-1,3 glucan on immune responses and disease resistance of healthy and aflatoxin B1-induced immunocompromised rohu (Labeo rohita Hamilton)

The immunostimulant β-1,3 glucan was fed at 0·1% in feed for 7 days to healthy and aflatoxin B₁(AFB₁)-induced immunocompromised fish, Labeo rohita (one of the major tropical carp species), in a 60 day trial. The effects of AFB₁, glucan and their interactions on non-specific and specific immunity levels and disease resistance of fish were studied. A single intraperitoneal injection of AFB₁at 1·25 mg kg⁻¹body weight) caused a significant (P< 0·05) reduction in non-specific immunity as measured through neutrophil phagocytic indices, serum bactericidal activity, and specific immunity as measured through bacterial agglutination titre against Edwardsiella tarda, as well as reduced protection against Aeromonas hydrophila challenge in comparison to control fish which were exposed neither to aflatoxin nor to glucan. Feeding of glucan to healthy fish raised the non-specific and specific immunity level and protection against bacterial infection compared with the control. Feeding of glucan to AFB₁-induced immunocompromised fish for 7 days significantly raised the degree of resistance against A. hydrophila challenge and the non-specific immunity level in comparison to non-treated AFB₁exposed fish. Although feeding of glucan was able to increase specific immunity, al measured through haemagglutination titre against sheep red blood cells, and bacterial (E. tarda) agglutination titre in healthy fish in comparison to all other groups, no significant increase in specific immunity to the aflatoxin-exposed group was seen.     

Molecular characterization and immunohistochemical localization of tilapia piscidin 3 in response to Aeromonas hydrophila infection in Nile tilapia

The antimicrobial activity of tilapia piscidin 3 (TP3) was determined in vitro against a locally isolated Aeromonas hydrophila. A 388 bp fragment was amplified from the TP3 cDNA and sequenced. The coding sequence (CDS) of TP3 was estimated to be 231 bp codes for 76 amino acids long and stop codon. In silico analysis was performed to detect both the signal peptide and the prodomain cleavage sites to follow the amino acids number 22 and 70, respectively. Based on this, a peptide 23 amino acids long with a remarkably high computed antimicrobial probability was synthesized and used in the subsequent experiments. The antimicrobial activity of TP3 was determined with minimum inhibitory concentration (MIC) and minim um bactericidal concentration (MBC) methods. TP3 exhibited relatively weak antimicrobial activities against the tested bacteria. A challenge experiment was then performed in Nile tilapia with low and high doses of A. hydrophila, followed by timely recognition; after 3, 6, 24 h, and 7 days of the specific TP3 gene expression, immunohistochemical localization was also performed. Histopathological examination revealed provoked inflammatory responses and congestion in the same organs of TP3 expression. Immunohistochemical localization showed that A. hydrophila induced tilapia fish to express TP3 after 24 h within the gills, intestine, hepatopancreas, spleen, and posterior kidney. In quantitative real time (RT)‐polymerase chain reaction analysis, the high dose showed higher mRNA expression levels than the low dose, and its expression levels increased in the A. hydrophila‐infected fish. It was therefore concluded that TP3 plays an essential role in fish immunity.     

Typing of <Emphasis Type="Italic">Aeromonas hydrophila</Emphasis> of fish and human diarrhoeal origin by outer membrane proteins and lipopolysaccharides

A study was undertaken to discriminate the strains of Aeromonas hydrophila isolated from fish and diarrhoeal samples by SDS-PAGE analysis of outer membrane proteins (OMPs) and lipopolysaccharides (LPSs). Common bands at 47 kDa positions for OMPs and at 31–38 kDa for LPSs were observed. No strain of A. hydrophila from clinical or fish samples was found identical in either OMPs or LPSs profile.