Production of superoxide anion by head-kidney leucocytes of Indian major carps immunised with bacterins of Aeromonas hydrophila

On contact with micro-organisms or any other antigenic particles which are recognised as phagocytosable, the leucocytes of vertebrates raise their oxygen consumption suddenly (respiratory burst). The consumed oxygen is then converted into reactive oxygen species (ROS). An effort has been made in the present study to estimate the production of superoxide anion, one of the ROS, by the resident as well as activated head-kidney leucocytes of Indian major carps. Activation was accomplished by immunisation with formalin killed (FK) and heat killed (HK) whole cell bacterins of Aeromonas hydrophila. In the ex vivo experiment conducted, activated leucocytes yielded a significant increase in O.D. values for nitroblue tetrazolium (NBT) reduction reaction, reflecting an increase in superoxide anion production, from day 1 post immunisation. Of the three species of Indian major carps immunised, Catla catla showed the greatest production of superoxide anion, followed by Labeo rohita and then by Cirrhinus mrigala. The enhancement of superoxide anion production of leucocytes by immunisation justifies the role of immunisation in the microbicidal defence mechanism of fish.     

Non-specific immune responses in juveniles of Indian major carps

The non‐specific immune parameters are useful to determine the health status of fish and to evaluate the immunomodulatory substances for fish farming as markers of pollution and disease resistance. Some of the important parameters, viz. superoxide production by neutrophils through nitroblue tetrazolium (NBT) assay; haemagglutination (HA), haemolysin (HLY) and bacterial agglutination titres; myeloperoxidase (MPO), and lysozyme activities, and alternative complement levels in serum of the juveniles of three Indian major carp species (Cirrhinus mrigala, Catla catla and Labeo rohita) were measured to establish their physiological normal range. A wide variation among the individuals within a species in the ranges of the most of the immune parameters was recorded. Significantly higher levels in the mean values of HA, HLY, bacterial agglutination titres; superoxide production by neutrophils in nitroblue tetrazolium assay; serum MPO and lysozyme activities, i.e. 371.20, 4.60, 18.80, 0.40, 0.62 and 6.55 μg ml^?1, respectively, were obtained in L. rohita except a much lower alternative haemolytic complement activity (29.06 units ml^?1) compared with the other two species. In most of the parameters, L. rohita showed the highest value, possibly indicating its more natural resistance compared with the other two species.     

Cloning, characterisation and expression of Aeromonas hydrophila major adhesin

Aeromonas hydrophila, an important pathogen in fish, is believed to cause diseases by adhesive and enterotoxic mechanisms. The adhesion is a prerequisite for successful invasion. In this study, the gene of a 43 kDa major adhesin (designated as AHA1) was cloned and expressed. Nucleotide sequence analysis of AHA1 revealed an open reading frame encoding a polypeptide of 373 amino acids with a 20-amino-acid putative signal peptide (molecular weight 40,737 Da). The amino acid sequences of Aha1p showed a very high homology with the other two outer membrane proteins of A. hydrophila. Using the T-5 expression system, this major adhesin Aha1p was expressed in Escherichia coli. The purified recombinant adhesin could competitively inhibit A. hydrophila from invading fish epithelial cells in vitro. Western-blot analysis showed that this major adhesin is a very conserved antigen among various strains of Aeromonas. When used to immunise blue gourami, the recombinant adhesin could confer significant protection to fish against experimental A. hydrophila challenge.     

CP7抗菌蛋白对嗜水气单胞菌的抑杀作用机理

[目的]研究多粘类芽孢杆菌(Paenibacillus polymyxa) CP7菌株的抗菌蛋白(CP7ACP)对嗜水气单胞菌的抑杀作用机理,为防治嗜水气单胞菌引起的鱼病提供新的潜在天然药物.[方法]采用抑菌试验、钼锑抗比色法和紫外光谱法研究其对嗜水气单胞菌S12菌株生长、磷泄漏和生物大分子的影响,并利用扫描电镜和透射电镜观察了嗜水气单胞菌细胞结构遭受的破坏作用.[结果]CP7ACP对嗜水气单胞菌的抑菌圈直径约8.1 mm,最小抑菌浓度(MIC)与最小杀菌浓度(MBC)分别为原液浓度的1/8和1/4;嗜水气单胞菌受CP7ACP处理后,电镜观察发现其细胞壁、细胞膜、细胞器以及菌体均受到不同程度的破坏,胞内的生物大分子和磷泄漏明显,基因组DNA发生增色效应.[结论]CP7ACP抑制嗜水气单胞菌生长,可用于防治嗜水气单胞菌引起的鱼病.     

Density-dependent growth performance of Indian major carps in rainwater reservoirs

The study was carried out to evaluate growth performance of Indian major carps at different stocking densities in rainwater reservoirs. In this experiment, the absolute growth performance of Catla catla, Labeo rohita and Cirrhinus mrigala at stocking densities of 5000, 8000 and 11 000 fingerlings per hectare was 377.7, 215.4 and 241.9; 370.2, 186.7 and 219.1; and 306.7, 163.4 and 180.6 g, respectively. The recorded biomass yield at 5000, 8000 and 11 000 fingerlings per hectare was 1035, 1572 and 1573 kg ha^?1 per 180 days of culture. The effect of stocking density on production performance (performance index, PI) was highly significant (P < 0.05) at the higher stocking density of 11 000 ha^?1, while there was no significant variation between PI at stocking densities of 5000 and 8000 ha^?1. This indicates optimum production performance at 8000 ha^?1, where yield is significantly higher (P < 0.05) than yield at 5000 ha^?1 and almost equal to the yield at 11 000 ha^?1. An increase in stocking density from 8000 to 11 000 ha^?1, however, showed a sharp decline in average mean body weight of each species, even with supplemental feeding. With an increase in stocking density, the biomass yield increased to an optimum (1572 kg ha^?1), with no substantial increase thereafter. Reductions in growth, which occurred at high density, did not appear to be due to poor water quality as the water quality did not differ significantly among various treatments. Thus, the reduced survival and growth at high density appears to be a behavioural interaction or physiological response to density itself.     

Uptake and processing of biofilm and free-cell vaccines of Aeromonas hydrophila in indian major carps and common carp following oral vaccination--antigen localization by a monoclonal antibody

Uptake and processing of biofilm (BF) and free-cell (FC) vaccines of Aeromonas hydrophila were studied in the Indian major carps catla Catla catla, and rohu Labeo rohita and in the common carp Cyprinus carpio following a single dose oral vaccination of 10(11) CFU g(-1) fish. Fish were sampled at 0.5, 1, 3, 6, 12, 24 h and at 2, 3, 5 and 10 d following vaccination and antigen localization was studied in the gut, kidney and spleen employing monoclonal antibody based immunofluorescence and immunoperoxidase. The general pattern of antigen localization was similar in catla, rohu and common carp. Initially, both the BF and FC antigens were localized in the gut lumen, followed by their uptake by intraepithelial vacuoles and macrophages. Antigen administered orally was also seen in the spleen and kidney. Both BF and FC antigens were detected in the gut lumen of carp within 30 min following oral delivery. However, BF antigen remained in the lumen of the hindgut for 48 h compared to 6 h in the case of FC antigen. In the early stages, BF antigen was localized in the gut epithelial vacuoles while FC antigen was associated with the small macrophages of the hindgut. Antigen localization in spleen and kidney was observed at 3 h and persisted even up to 10 d following oral delivery. In general, there was a distinct difference between BF and FC vaccines in the duration of retention and quantity of uptake in the gut, kidney and spleen.     

Comparative and evolutionary analysis of mitochondrial genes in Indian major carps

Direct sequencing of mitochondrial DNA regions such as cytochrome b, ATPase 6/8 and control region was performed to study comparative and evolutionary status of the three mitochondrial genes in Labeo rohita, Catla catla and Cirrhinus mrigala. DNA sequence alignment among species using specific software revealed comparative rates of divergence with considerably faster and more heterogeneous substitution rate for control region as compared to cytochrome b and ATPase 6/8. Despite the relatively high variability of control region, the overall levels of sequence divergence were low in coding regions. Two protein coding genes and the control region with varying degree of sequence divergence established two distinct groups which are genetically distant from each other exhibiting identical phylogenetic structure in IMCs. Closest relationship was between Labeo rohita and Catla catla indicating that they might have diverged from a common ancestral stock in genealogical lineage whereas Cirrhinus mrigala showed greater divergence with all the three DNA regions studied. Findings of this study will help to understand evolution of mitochondrial DNA genes in carps and facilitate future investigations on phylogeographic structure of Indian carps.     

Production of cholera-like enterotoxin by Aeromonas hydrophila

A total of 249 strains of mesophilic Aeromonas including 179 A. hydrophila and 70 A. caviae were tested for production of cholera-like enterotoxin by reversed passive latex agglutination (RPLA) assay. A cholera-like enterotoxin neutralized with cholera antitoxin was demonstrated in the culture filtrates from eight (4.5%) of the 179 A. hydrophila strains, while none of A. caviae strains revealed the enterotoxin production in the test. Production of the cholera-like enterotoxin in the eight strains of A. hydrophila was also confirmed by enzyme-linked immunosorbent assay (ELISA).     

Family association between immune parameters and resistance to Aeromonas hydrophila infection in the Indian major carp, Labeo rohita

Seven innate immune parameters were investigated in 64 full-sib families (the offspring of 64 sires and 45 dams) from two year-classes of farmed rohu carp (Labeo rohita). Survival rates were also available from Aeromonas hydrophila infection (aeromoniasis) recorded in controlled challenge tests on a different sample of individuals from the same families. Due to strong confounding between the animal additive genetic effect and the family effects (common environmental + non-additive genetic), reliable additive (co)variance components and hence heritabilities and genetic correlations could not be obtained for the investigated parameters. Therefore, estimates of the association of challenge test survival with the studied immune parameters were obtained as product moment correlations between family least square means. These correlations revealed statistically significant (p < 0.05) negative correlations of survival with bacterial agglutination titre (−0.48), serum haemolysin titre (−0.29) and haemagglutination titre (−0.34); and significant positive correlation with ceruloplasmin level (0.51). The correlations of survival to aeromoniasis with myeloperoxidase activity, superoxide production and lysozyme activity were found to be not significantly different from zero (p > 0.05). Assuming that the negatively correlated candidate traits are not favourable as indirect selection criteria, the results suggest that ceruloplasmin level could potentially be a marker for resistance to aeromoniasis in rohu. The use of this immune parameter as an indirect selection criterion for increased resistance to aeromoniasis in rohu will, however, require that the parameter shows significant additive genetic variation and a significant genetic correlation with survival. Further studies are therefore needed to obtain a reliable heritability estimate for ceruloplasmin and its genetic correlation with survival from aeromoniasis.     

Reduction of diverse electron acceptors by Aeromonas hydrophila

Aeromonas hydrophila ATCC 7966 grew anaerobically on glycerol with nitrate, fumarate, Fe(III), Co(III), or Se(VI) as the sole terminal electron acceptor, but did not ferment glycerol. Final cell yields were directly proportional to the amount of terminal electron acceptor provided. Twenty-four estuarine mesophilic aeromonads were isolated; all reduced nitrate, Fe(III), or Co(III), and five strains reduced Se(VI). Dissimilatory Fe(III) reduction by A. hydrophila may involve cytochromes. Difference spectra obtained with whole cells showed absorption maxima at wavelengths characteristic of c-type cytochromes (419, 522, and 553 nm). Hydrogen-reduced cytochromes within intact cells were oxidized by the addition of Fe(III) or nitrate. Studies with respiratory inhibitors yielded results consistent with a respiratory chain involving succinate (flavin-containing) dehydrogenase, quinones and cytochromes, and a single Fe(III) reductase. Neither anaerobic respiration nor dissimilatory metal reduction by members of the genus Aeromonas have been reported previously. Received: 24 June 1997 / Accepted: 20 October 1997     

Characterization of lactoferrin binding by Aeromonas hydrophila.

Various lactoferrin preparations (iron-saturated and iron-depleted human milk lactoferrins and bovine milk and colostrum lactoferrins) were bound by Aeromonas hydrophila. Binding was (i) reversible (65% of bound lactoferrin was displaced by unlabeled lactoferrin), (ii) specific (lactoferrin but not other iron-containing glycoproteins such as ferritin, transferrin, hemoglobin, and myoglobin inhibited binding), and (iii) significantly reduced by pepsin and neuraminidase treatment of the bacteria. The glycosidic domains of the lactoferrin molecule seem to be involved in binding since precursor monosaccharides of the lactoferrin oligosaccharides (mannose, fucose, and galactose) and glycoproteins which have homologous glycosidic moieties similar to those of the lactoferrin oligosaccharides (asialofetuin or fetuin) strongly inhibited lactoferrin binding. A. hydrophila also binds transferrin, ferritin, cytochrome c, hemin, and Congo red. However, binding of these iron-containing compounds seems to involve bacterial surface components different from those required for lactoferrin binding. Expression of lactoferrin binding by A. hydrophila was influenced by culture conditions. In addition, there was an inverse relationship between lactoferrin binding and siderophore production by the bacterium.     

Characterization of a pilus produced by Aeromonas hydrophila

A pilus produced by Aeromonas hydrophila was purified and partially characterized. The pilin monomers had an apparent molecular weight of 17,000. Agglutination studies indicated serological cross-reactivity in the pili of A. hydrophila strains. Presence of pili did not correlate with hydrophobicity or haemagglutinating ability of the bacteria.     

Characterization of lactoferrin binding by Aeromonas hydrophila.

Various lactoferrin preparations (iron-saturated and iron-depleted human milk lactoferrins and bovine milk and colostrum lactoferrins) were bound by Aeromonas hydrophila. Binding was (i) reversible (65% of bound lactoferrin was displaced by unlabeled lactoferrin), (ii) specific (lactoferrin but not other iron-containing glycoproteins such as ferritin, transferrin, hemoglobin, and myoglobin inhibited binding), and (iii) significantly reduced by pepsin and neuraminidase treatment of the bacteria. The glycosidic domains of the lactoferrin molecule seem to be involved in binding since precursor monosaccharides of the lactoferrin oligosaccharides (mannose, fucose, and galactose) and glycoproteins which have homologous glycosidic moieties similar to those of the lactoferrin oligosaccharides (asialofetuin or fetuin) strongly inhibited lactoferrin binding. A. hydrophila also binds transferrin, ferritin, cytochrome c, hemin, and Congo red. However, binding of these iron-containing compounds seems to involve bacterial surface components different from those required for lactoferrin binding. Expression of lactoferrin binding by A. hydrophila was influenced by culture conditions. In addition, there was an inverse relationship between lactoferrin binding and siderophore production by the bacterium.     

RAPD analysis of Aeromonas salmonicida and Aeromonas hydrophila

The randomly amplified polymorphic DNA (RAPD) technique was used to analyse the genetic differentiation of 13 strains of Aeromonas salmonicida subsp. salmonicida , and seven strains of Aer. hydrophila. Reproducible profiles of genomic DNA fingerprints were generated by polymerase chain reaction (PCR) using a single randomly designed primer. The RAPD profiles of all the non-motile aeromonads, Aer. salmonicida subsp. salmonicida were identical. However, profiles of the motile aeromonads, Aer. hydrophila differed between isolates. These findings reveal genomic homogeneity in Aer. salmonicida subsp. salmonicida and genetic variety in Aer. hydrophila strains.     

Electroporation-mediated transformation of Aeromonas hydrophila

A strain of Aeromonas hydrophila producing copolyesters of 3-hydroxybutyrate and 3-hydroxyhexanoate, abbreviated as PHBHHx, was successfully transformed by electroporation. The plasmid used was a broad host range plasmid pBBR1MCS. Electroporation conditions were varied systemically to develop an electroporation protocol. The optimal yield of transformant was approximately 4x10(2) CFU/microg DNA at 12.5 kV/cm and 1000 Omega, resulting in a time constant of approximately 5 ms. The A. hydrophila transformants expressed plasmid-encoded resistance to chloromphenicol. Plasmid DNA in the A. hydrophila transformant was stably maintained. This is the first report of transformation of bacteria A. hydrophila.     

A study of the antibacterial mechanism of pinocembrin against multidrug-resistant Aeromonas hydrophila

International Microbiology - Aeromonas hydrophila is a common pathogen in fish that has caused severe economic losses in aquaculture worldwide. With the emergence of bacterial resistance, it is...     

Enhancement of protective immunity in European eel (Anguilla anguilla) against Aeromonas hydrophila and Aeromonas sobria by a recombinant Aeromonas outer membrane protein

To develop a vaccine, which can simultaneously prevent the diseases caused by various pathogenic bacteria in fish, we try to find a conserved outer membrane protein (OMP) antigen from different bacterial pathogens. In this study, an OMP fragment of 747 bp (named as Omp-G), which was highly conserved in seven Aeromonas OMP sequences from the NCBI database, was amplified by PCR from one Aeromonas sobria strain (B10) and two Aeromonas hydrophila strains (B27 and B33) with the designed specific primers. The sequence was cloned into pGEX-2T (6 × His-tag) vector, expressed in Escherichia coli system, and then the recombinant protein (named as rOmp-G) was purified with nickel chelating affinity chromatography. The purified rOmp-G showed a good immunogenicity in rabbits and well-conserved characteristics in these three pathogens by enzyme-linked immunosorbed assay. Furthermore, the rOmp-G also showed good immunogenicity in eels (Anguilla anguilla) for eliciting significantly increased specific antibodies (P < 0.01), and providing higher protection efficiencies (P < 0.05) after the pathogens challenge. The values of the relative percent survival in eels were 70% and 50% for two A. hydrophila strain challenge, and 75% for A. sobria strain challenge. This is the first report of a potential vaccination in eels that simultaneously provide protectiveness against different Aeromonas pathogens with a conserved partial OMP.     

Optional production and utilization of polysaccharide by Aeromonas hydrophila

Glucans from the fish pathogen Aeromonas hydrophila have been extracted andpurified by a method utilizing phenol/water followed by sodium deoxycholate rather than the traditional sodium hydroxide extraction. Presence of substantial amounts of these glucans was shown to be dependant on whether or not the substrate contained dextrose, a point which had import because of the low carbohydrate environment in which this species must survive and multiply. These glucans, produced in the log phase, were utilized during the later growth period.The structures of the two purified glucans were examined by methylation analysis, periodate oxidation, and enzymatic degradation. The results indicated that A. hydrophila under low-carbohydrate growth conditions produced two similar but distinguishable 14 linked glucans substituted 16 by single monosaccharide residues or short chains to give an amylopectinglycogen type of polysaccharide.