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Effect of temperature and glucose concentration on the N-butanoyl-L-homoserine lactone production by Aeromonas hydrophila.

N-butanoyl-L-homoserine lactone (C4-HSL) production by Aeromonas hydrophila 519 has been established. C4-HSL production at 22 and 30 degrees C was found after 20-24 h of incubation corresponding to a population density of ca. 10(8)-10(9) cfu/ml, respectively. Reduced C4-HSL production was noted after 72 h of incubation at 12 degrees C when the culture reached ca. 10(9) cfu/ml. No C4-HSL production was detected at 37 degrees C, although a dense population was obtained. In LB broth supplemented with 0.1% and 0.5% glucose, C4-HSL production was noted whereas with 1% glucose no C4-HSL was detected although a high colony count was obtained. In the latter culture residual levels of glucose (0.65%) were found after 43 h whereas in the 0.1% and 0.5% supplemented LB, glucose was quickly consumed which may have stimulated C4-HSL production. In conclusion, the present study shows an effect of environmental conditions (temperature, glucose concentration) on the C4-HSL production and warrants further investigation to elucidate the effect of external conditions on production of AHL signal molecules to reveal the relevance of quorum sensing in, e.g. foods.

4-Butyrolactone↗

Antigen expression in biofilm cells of Aeromonas hydrophila employed in oral vaccination of fish.

Total protein, S-layer protein and lipopolysaccharides (LPS) of biofilm cells of Aeromonas hydrophila were analysed by SDS-PAGE and compared with that of planktonic cells. In the whole cell lysate of biofilm cells, about 15 proteins were repressed while three new proteins were expressed compared to that in planktonic cells. Interestingly, in biofilm cells the S-layer proteins were lost and LPS showed an additional high molecular weight band compared to that in planktonic cells. We propose that the change in LPS profile must have contributed to the loss of S-layer. Also, the high molecular weight band of LPS might play a role in the better performance of biofilm oral vaccine by eliciting a protective immune response.

Aeromonas hydrophila↗

The effect of various Aeromonas bestiarum vaccines on non-specific immune parameters and protection of carp (Cyprinus carpio L.).

Aeromonas bestiarum is one of the causal agents of motile aeromonad infection/motile aeromonad septicemia (MAI/MAS) in fish. Infections of the bacterium is an increasing problem in commercial carp (Cyprinus carpio L.) farmed in Poland. Non-specific immune response of the fish, vaccinated with oil-emulsified experimental vaccines containing formalin killed whole cells (WCs), formalin killed whole culture (WCt) or crude LPS (50 or 1250 microg per fish) were studied on days 7 and 30 after vaccination. Fish vaccinated for 30 days were challenged with the pathogen and mortalities recorded over 14 days. The cumulative mortalities were 10%, 0%, 20% and 20% in WCs, WCt, LPS-1250 and LPS-50 groups, respectively, whereas 70% fish died in the control group. Vaccinated fish showed significant increase of phagocytic activity (PA) and phagocytic index (PI). The total serum Ig (TSIg) level was significantly higher in most vaccinated fish groups than in control. Moreover, WCs and WCt induced significant increase of mucus lysozyme level in vaccinated fish.

Aeromonas↗

Evaluation of biofilm of Aeromonas hydrophila for oral vaccination of Clarias batrachus--a carnivore model.

Biofilm of Aeromonas hydrophila was evaluated for oral vaccination of walking catfish (Clarias batrachus L.). Fish were fed with fish paste incorporating biofilm (BF) or free cells (FC) of A. hydrophila for 20 days and monitored for serum antibody production up to 60 days post-vaccination. Serum agglutinating antibody titre and relative percent survival (RPS) following challenge were found to be significantly higher in catfish fed with BF vaccine compared to that with FC.

Administration, Oral↗

Modulation of the immune response to an Aeromonas hydrophila aroA live vaccine in rainbow trout: effect of culture media on the humoral immune response and complement consumption.

The Aeromonas hydrophila aroA is an attenuated strain that has been assessed as a live vaccine in rainbow trout, Oncorhynchus mykiss. In this study the effects of different culture media used to grow the strain on its survival after in vitro exposure to rainbow trout serum, and on its immunogenicity in rainbow trout were compared. Four culture media were tested: Luria broth (LB), Luria broth with 0.25% glucose, trypticase soy broth (TSB), and brain-heart infusion broth (BHIB). Bacteria grown in culture media with glucose (TSB, BHIB and LB with 0.25% glucose) showed reduced complement consumption and a lower serum susceptibility. O. mykiss vaccinated with inocula prepared with BHIB- and LB-grown aroA cells resuspended in phosphate-buffered saline (PBS) showed higher and longer-lasting serum agglutinating antibody titres than those vaccinated with TSB-grown bacteria. Thus, a direct relationship between serum resistance and immunogenicity could not be established, but BHIB and LB culture media were the most effective in increasing the immunogenicity of the A. hydrophila aroA vaccine.

Aeromonas hydrophila↗

Protection against experimental Aeromonas salmonicida infection in carp by oral immunisation with bacterial antigen entrapped liposomes.

Liposome-entrapped atypical Aeromonas salmonicida antigen was prepared to investigate the potential protective efficacy for A. salmonicida infection. Carp (Cyprinus carpio) were immunised orally with liposome-entrapped A. salmonicida antigen. After immunisation, significantly higher antigen-specific antibodies were detected in serum, intestinal mucus and bile than non-immunised control group. Furthermore, immunised carp were challenged by immersion with 1 x 10(6) cfu ml(-1) of A. salmonicida for 60 min. Of the eight non-immunised carp, three carp died (62.5% survival), whereas five out of six (83.5%) immunised survived. Furthermore, the development of skin ulcers was significantly inhibited in carp immunised with liposomes containing A. salmonicida antigen. These results suggest that liposomes containing A. salmonicida antigen have the potential for the induction of a protective immune response against atypical A. salmonicida infection and also suggest the possibility of developing a vaccine that may ultimately be used for the prevention of fish diseases.

Administration, Oral↗

Expressed sequence tags analysis of Atlantic halibut (Hippoglossus hippoglossus) liver, kidney and spleen tissues following vaccination against Vibrio anguillarum and Aeromonas salmonicida.

To investigate the response of Atlantic halibut to vaccination and pathogen exposure, a cDNA library was constructed from liver, kidney and spleen mRNA collected following vaccination against Vibrio anguillarum and Aeromonas salmonicida. After sequencing 1114 clones 1072 (96.23%) readable sequences were obtained of which 106 sequences are the first reported from the fish. Of these, 182 clones (16.98%) contained cell/organism defence genes including immunoglobulin light chain, MHC class I and II, interferon consensus sequence binding protein, B-cell receptor-associated protein, early B-cell factor, 10 complement components, heat shock protein 70 and 90, antimicrobial peptides hepcidin type 1 and 2, and CC chemokine (macrophage inflammatory protein-1 beta-like chemokine, MIP-1beta). Expression of MIP-1beta-like was elevated in the kidney and spleen at 1, 2, 7 and 14 days post vaccination. Functional genes involved in cellular processes of hematopoietic tissues were also identified. These results indicate that this cDNA library contains many important genes involved in the immune response, making it an important resource for studying the response of Atlantic halibut to vaccination or pathogen exposure.

Aeromonas salmonicida↗

Evaluation of DNA vaccination of spotted sand bass (Paralabrax maculatofasciatus) with two major outer-membrane protein-encoding genes from Aeromonas veronii.

Genes encoding two major outer membrane proteins (OMPs) of the bacterial pathogen Aeromonas veronii, Omp38 and Omp48, were used to construct DNA vaccines. The protective effect of such vaccines against motile aeromonad septicaemia was evaluated in spotted sand bass (Paralabrax maculatofasciatus), an endemic species of the Mexican Northwest Pacific coast and a potential resource for the aquaculture industry. Weak protein expression, as determined by immunoblotting, was observed after transfection of eukaryotic cells with the DNA vaccines. Fish immunized with a single intramuscular injection of 20 microg of the omp38 and omp48 DNA vaccines showed slightly, but significantly elevated serum antibody levels 4 and 6 weeks after vaccination, compared to fish vaccinated with the control plasmid pcDNA3.1. Spotted sand bass vaccinated with the omp38 and omp48 DNA vaccines and challenged with A. veronii by intraperitoneal route recorded a relative percent survival (RPS) between 50 and 60%. Histopathological signs of motile aeromonad septicaemia were observed in around 40% of omp38 and omp48-vaccinated fish and 80% of pcDNA3.1-vaccinated control fish. The results indicate that P. maculatofasciatus vaccinated with a single dose of DNA plasmids encoding the major OMPs from A. veronii shows partial protection against infection and mortality by A. veronii experimental infection.

Aeromonas↗

Administration of yeast glucan enhances survival and some non-specific and specific immune parameters in carp (Cyprinus carpio) infected with Aeromonas hydrophila.

Effects of beta-glucan administration on survival and immune modulations were studied in Cyprinus carpio against the bacterial pathogen, Aeromonas hydrophila. Beta-glucan was extracted from Saccharomyces cervisiae and purified. A virulent strain of the pathogen A. hydrophila was collected from infected fish. Different concentrations of beta-glucan were administered to test animals on day 1, 3 and 5 through different routes (intraperitoneal injection (ip), bathing and oral administration). Control and test animals were challenged by ip injection of LD50 concentration of A. hydrophila on day 7 and mortality was observed and Relative Percent Survival (RPS) was calculated. Intraperitoneal injection of 500 microg of glucan significantly enhanced the RPS; bathing and oral administration of glucan did not influence the RPS. On day 7, test animals injected with 100, 500 and 1000 microg of glucan had a significant increase in total blood leucocyte counts and an increase in the proportion of neutrophils and monocytes. Superoxide anion production by kidney macrophages was also elevated. RT-PCR and northern blot analysis of interleukin-1 mRNA showed elevated expression in kidney on day 7 in fish injected with glucan. Glucan had an adjuvant effect on antibody production as pretreatment by injection of 100-1000 microg glucan/fish resulted in the highest antibody titer against A. hydrophila following vaccination. Classical and alternative complement pathways were not affected by glucan administration by any of the three routes.

Aeromonas hydrophila↗

The contribution of Aeromonas salmonicida extracellular products to the induction of inflammation in Atlantic salmon (Salmo salar L.) following vaccination with oil-based vaccines.

Intraperitoneal injection of Atlantic salmon with oil-based vaccines often results in severe side effects. Aeromonas salmonicida subspecies salmonicida, a primary antigen in the vaccines, produces extracellular products (ECPs) that are included in the formulation but the role of ECPs in inducing side effects is not well understood. In the present study, we evaluated the contribution of ECPs to early inflammatory reactions since early events determine the outcome of inflammation. Five groups of Atlantic salmon pre-smolts were injected intraperitoneally with one of the following preparations: (1) A. salmonicida water-in-oil (w/o) containing standard amounts of ECPs; (2) A. salmonicida (w/o) with ECPs concentrated five times; (3) A. salmonicida (w/o) without ECPs (ECPs were removed by washing and re-suspension of the bacteria prior to formulation); (4) w/o only (without antigens), and (5) physiological saline. Tissue sections of the injection site (pyloric caeca and surrounding areas) were collected at monthly intervals for 4 months in phosphate buffered formalin and processed for light microscopy. Computer-assisted microscopy with the help of Image Pro analysis program was used to measure the area of inflammation on H&E stained sections. Differential cell counts of leucocytes involved in the inflammatory reaction were also done based on morphology. Overall results show that fish injected with vaccines containing concentrated amounts of ECPs displayed a higher average area of inflammation compared to all other groups. In contrast, washed preparations induced mild reactions compared to vaccines containing either standard or concentrated ECPs. Mild, non-persistent reactions were observed in the group injected with oil adjuvant only. Neutrophils were persistent in inflammations induced by all preparations except w/o only. No inflammatory reactions were observed in the group injected with PBS. The results suggest that ECPs are pro-inflammatory in Atlantic salmon. It is anticipated that ECPs are more readily exposed to inflammatory cells than the bacterial cells themselves during early stages of inflammation because of their orientation at the water-oil interface. The results indicate that ECPs of A. salmonicida play an important role in the induction of early inflammatory reactions. It is also documented that the combination of antigens with oil adjuvants, and not the adjuvants alone, is the inducer of strong inflammatory reactions in Atlantic salmon.

Aeromonas salmonicida↗

Dietary administration of bovine lactoferrin influences the immune ability of the giant freshwater prawn Macrobrachium rosenbergii (de Man) and its resistance against Aeromonas hydrophila infection and nitrite stress.

Haemocyte count, phenoloxidase activity, agglutinin levels, total protein content, bacterial clearance efficiency, resistance to the pathogen Aeromonas hydrophila and nitrite stress were measured in the giant freshwater inter-moult sub-adult prawn Macrobrachium rosenbergii (15-20 g) which had been fed diets containing bovine lactoferrin (Lf) at 50, 100, 200mg kg(-1) feed for 7 or 14 days. M. rosenbergii fed a diet containing 100mg Lf kg(-1) diet for 7 days showed significant (P<0.05) increase in total protein levels, agglutination titres against bacteria A. hydrophila and rabbit RBC, phenoloxidase activity, bacterial clearance (as observed through reduced number of circulating bacteria) as well as survival against A. hydrophila challenge. Increased bacterial clearance was also noticed in prawns fed Lf at 50 or 200mg kg(-1) for 14 days compared to control. Feeding of Lf at 50mg kg(-1) diet for 7 or 14 days was able to enhance only PO activity and reduce percent mortality against A. hydrophila challenge compared to its control. Total haemocyte count was higher in the lowest dose of Lf feeding, i.e. 50mg kg(-1) for 7 days. However, there was no significant alteration in the differential haemocyte population with respect to graded levels of Lf feeding for 7 or 14 days. A notable reduction in mortality percent after 120 h of nitrite stress was observed in prawn fed Lf at 100mg kg(-1) diet for 14 days. On the contrary, feeding of the highest dose of Lf, i.e. 200mg kg(-1) diet for 14 days failed to stimulate most of the innate immune parameters or reduce the percent mortality against A. hydrophila challenge or nitrite stress. It is therefore concluded that administration of Lf in the diet at 100mg kg(-1) for 7 days could enhance the immune ability of M. rosenbergii and increase its resistance to A. hydrophila infection or nitrite stress.

Aeromonas hydrophila↗

Effect of Mangifera indica kernel as a feed additive on immunity and resistance to Aeromonas hydrophila in Labeo rohita fingerlings.

The study evaluated the efficacy of dietary doses of Mangifera indica (mango) kernel on the immune response and disease resistance of Labeo rohita fingerlings against the bacterial pathogen Aeromonas hydrophila infections in. L. rohita fingerlings fed diet containing 0 (Control), 1g, 5 g, 10 g mango kernel kg(-1) dry diet for 60 days. Biochemical (serum total protein, albumin, globulin, albumin:globulin ratio, blood glucose), haematological (WBC, RBC, haemoglobin content) and immunological (superoxide anion production, lysozyme, serum bactericidal activity) parameters of fish were examined at 20, 40 and 60 days of feeding. Fish were challenged with A. hydrophila 60 days post feeding and mortalities were recorded over 10 days post-infection. The results demonstrate that fish fed with mango kernel showed enhanced superoxide anion production, lysozyme, serum bactericidal, serum protein, albumin (P<0.05) compared with the control group. The mortality (%) was recorded up to 10 th day post-challenge. Less survivability was observed in control group (50%) up to day 10 after infection. The survivability was higher in experimental diets. The group fed 5 g kernel kg(-1) dry diet showed highest percentage survival (98%). These results indicate that mango kernel stimulates the immunity and makes L. rohita more resistant to A. hydrophila infection.

Aeromonas hydrophila↗

In vivo humoral and cellular reactions, and fate of injected bacteria Aeromonas hydrophila in freshwater prawn Macrobrachium rosenbergii.

Live non-opsonized and opsonized Aeromonas hydrophila were injected into juveniles of freshwater prawn Macrobrachium rosenbergii to study the cells involved in phagocytosis, distribution of bacteria, cellular reactions and clearance of both forms of bacteria from the system. The bacteria were rapidly distributed to various tissues viz., gills, heart, hepatopancreas within 1h, and the tissues revealed haemocytic nodule formation after 3 h of injection. There was rapid clearance of both the forms of bacteria from the circulation. However, clearance efficiency was significantly (P < 0.05) faster in the case of opsonized bacteria at 12 h after injection. Similarly, the nodule formation, that was prominent in cardiac musculature, was rapidly eliminated from the tissues of the group injected with opsonized bacteria as compared to non-opsonized bacteria injected group, thus confirming the existence of opsonic factors in haemolymph of this prawn. In another experiment, various dose levels of bacteria were injected intramuscularly into prawns and haemolymph was collected after 1, 6, 24, 72 h and 7 days of injection to study various immune parameters. Although, no major alterations in the total and differential haemocyte counts were observed in bacteria injected prawns compared to control, there was a significant decline in phenoloxidase activity in the highest dose bacteria injected group at the earlier phase and a rise in agglutinin levels at the later phase of the experimental period in the higher dose groups.

Aeromonas hydrophila↗

Development and identification of KASP-SNP markers correlated with Aeromonas hydrophila resistance traits in blunt snout bream (Megalobrama amblycephala).

The blunt snout bream (Megalobrama amblycephala) is an economically important freshwater fish species. However, it is highly susceptible to Aeromonas hydrophila infection, especially in intensive pond aquaculture in China. Molecular marker-assisted selection provides an efficient approach for breeding disease-resistant varieties; however, the key genes or molecular markers linked to A. hydrophila resistance remain scarce in this species. A 436 differential SNP sites with disease-resistant were screened on basis of whole-genome resequencing. Then, a high-throughput genomic KASP genotyping technique was utilized to discover favorable genes and SNP sites associated with A. hydrophila resistance. A total of 46 KASP markers were successfully developed with an accuracy of 92&#xa0;%. These markers were used to genotyping 120 blunt snout bream individuals. Through trait correlation analysis and general linear models (GLM), five SNPs significantly (P&#xa0;<&#xa0;0.05) associated with resistance to A. hydrophila were identified and mapped to five candidate genes (btnl2, cfhr2, slc47a1, neu3, nlrp1). Survival rate of individuals carrying the dominant genotype demonstrated an average survival rate of 81.39&#xa0;%, which represents a 69.35&#xa0;% increase in comparison with that of 48&#xa0;% in total population. This effect was validated in an external population of 100 fish. These findings identify key genetic markers associated with A. hydrophila resistance and provide a direction for elucidating the underlying molecular immune mechanisms, thus establishing a genetic foundation for future breeding strategies.

Cyprinidae↗

Genomic characterization of a hypervirulent Aeromonas veronii NN0115 from Nile tilapia and head kidney transcriptome of infected fish reveals B-cell-dominated immune response with specific immunoglobulin downregulation.

Aeromonas veronii is a pathogen of multiple fish species, yet systematic understanding of its infection in Nile tilapia (Oreochromis niloticus) remains limited. A dominant strain, NN0115, was isolated from a natural outbreak and identified as A. veronii by 16S rRNA and whole-genome average nucleotide identity (ANI, 96.33%). Experimental infection revealed high virulence (LD50&#x202f;=&#x202f;3.41&#x202f;&#xd7;&#x202f;106&#x202f;CFU/mL, equivalent to 8.53&#x202f;&#xd7;&#x202f;104&#x202f;CFU/fish). The genome is 4.58&#x202f;Mb (58.57% GC) and encodes 4216 proteins. Virulence factor analysis identified 1253 genes, dominated by motility-related (264) and immune modulation (208) factors. Genomic island GI2 harbors 7 virulence genes and two dual-function resistance-virulence genes. The strain is resistant to 9 of 25 agents tested but carries three RND efflux pump genes whose predicted resistance was not phenotypically observed. The head kidney transcriptome of tilapia at 24&#x202f;h post-bacterial infection identified 773 differentially expressed genes; among them, 57 were immunoglobulin (Ig) genes, and 56 were down-regulated. Integration of published single-cell transcriptomic data showed that non-Ig B-cell marker genes were down-regulated by 32%, whereas Ig genes were reduced by 63%, indicating selective transcriptional suppression of Ig genes rather than a general decrease in B-cell transcriptional activity. Together, this study provides a comprehensive characterization of a highly virulent A. veronii from Nile tilapia and reveals that selective downregulation of B-cell Ig genes is the dominant transcriptional feature of the host head kidney response.

Animals↗

Predictive modelling of growth and measurement of enzymatic synthesis and activity by a cocktail of selected Enterobacteriaceae and Aeromonas hydrophila.

The possibility was examined of developing a predictive model that would predict food spoilage by combining microbial growth (increase in cellular number) with extracellular enzymatic activity of a cocktail of five strains of Enterobacteriaceae: Escherichia coli, Enterobacter agglomerans, Klebsiella oxytoca, Klebsiella pneumoniae and Proteus vulgaris and one Aeromonas hydrophila strain. Estimations of growth and enzyme activity were made within a three-dimensional matrix of conditions: temperature 2-20 degrees C, pH value 4.0-7.5 and water activity (a(w)) 0.95-0.995. A mathematical model was constructed which predicted growth based on increases in cell number. However, although notable effects of extracellular lipases and proteases were detected, it was not possible to model enzymatic activity and prepare a combined model because the data did not follow the characteristic profile that would allow curve-fitting. Nevertheless, the model for microbial growth and information relating to enzyme activity will be made freely available in a database on the internet.

Aeromonas hydrophila↗

An investigation of the Gamma hypothesis: a predictive modelling study of the effect of combined inhibitors (salt, pH and weak acids) on the growth of Aeromonas hydrophila.

The Gamma hypothesis, that multiple inhibitory factors combine independently, is the underlying hypothesis for the quantification of the Hurdle concept used in food manufacture. The literature, however, is confused as to whether interactive effects exist and under which circumstances they occur, if at all. Using the method of time to detection (TTD), the inhibitory effect of pH, salt and specific weak acids (acetic, propionic, sorbic and benzoic) and combinations of these with respect to the growth of Aeromonas hydrophila (ATCC 7966) were analysed. A model based on the relative rate to detection described all combinations analysed as having independent effects on the TTD. No synergistic interactions were found between pH and salt, between pH and individual weak acids or between combinations of weak acids and pH for any of the systems under study. This study supports the validity of the Gamma concept -- that individual environmental effects act independently and should, in turn, facilitate attempts to model the growth of other microorganisms under a variety of conditions.

Acids↗

Placental and intestinal alkaline phosphatases are receptors for Aeromonas sobria hemolysin.

The Aeromonas sobria hemolysin causes diarrhea following infection by this enteropathogenic bacterium. We previously identified the putative receptor for A. sobria hemolysin as a p66 protein on Intestine 407 cells (Microb. Pathog. 27 (1999) 215-221). Here, we have partially purified and obtained a peptide mass fingerprint of p66 which revealed its identity with placental alkaline phosphatase (PLAP). Recombinant PLAP expressed in 293T cells was also found to bind to hemolysin and the binding was found not to be dependent on the N-linked glycosylation of PLAP. By immunohistochemical analysis, PLAP expression was detected in human intestinal mucosa, the target tissue in disease. In addition to PLAP, hemolysin also binds to intestinal alkaline phosphatase (IAP), an enzyme that is also abundantly expressed in intestine. Thus, both PLAP and IAP are very likely involved in the pathogenesis of diarrhea caused by this bacterial toxin.

Aeromonas↗