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.     

Is Aeromonas hydrophila the dominant motile Aeromonas species that causes disease outbreaks in aquaculture production in the Zhejiang Province of China?

The significance of Aeromonas hydrophila in association with disease outbreaks in aquaculture production in the Zhejiang province of China was investigated. Bacteriological examination of moribund fish and crabs resulted in 95 bacterial isolates: 88 bacterial isolates from fish and 7 isolates from crabs. PCR and traditional biochemical methods were used for identification of A. hydrophila. Out of 69 motile aeromonads, 35 isolates were identified as A. hydrophila by biochemical tests. However, 6 of those were not identified as A. hydrophila by a species specific PCR method. Serotyping revealed 2 dominant serotypes (O9 and O97) among A. hydrophila isolates. The data presented show that approximately 42% of the motile aeromonads isolated from disease outbreaks among various fish species were A. hydrophila. It is noteworthy that A. hydrophila accounted for more than 50% of the isolated aeromonands isolated from crucian carp Carassius carassius and Wuchang bream Megalobrama amblycephala with haemorrhagic septicaemia. Although this species was the most frequently isolated organism from internal organs of diseased fish and crabs in the present study, other motile Aeromonas spp. were also found. The PCR assay was useful in preventing misidentification of A. hydrophila, which may occur when only phenotypic tests are employed.     

A preliminary study of parasites and diseases of perch in an intensive culture system

The recent development of intensive rearing of European perch Perca fluviatilis in warm water effluents revealed important pathological problems. This study gives a preliminary overview of parasites and diseases encountered in experimental perch farming. No virus has been recorded since the beginning of the experiments in 1993. By contrast, mass mortality caused by bacterial disease alone or associated with other pathogens were frequently observed. The main identified bacterial species were: Aeromonas sp., A. veronii, A. hydrophila, Streptococcus sp., Staphylococcus sp., Vibrio fluviatilis and Enterobacter agglomerans. Protozoans were the most common parasites observed on cultured P. fluviatilis. High mortality rate due to Ichthyobodo necator and Trichodina sp. may take place at different stages of perch rearing. In contrast, the infestation by Ambiphrya sp. and Heteropolaria sp. was size- or age-related; these two ciliates infested perch weighing < 1 g and > 20 g, respectively. In spite of the presence of other parasites such as Ichthyophthirius multifiliis, Hexamita spp., Gyrodactylus sp. and Argulus sp. in the rearing system P. fluviatilis does not appear to be affected. Further abnormalities or diseases inherent to intensive culture systems are also described.     

Bacteriocin-like substance (BLS) production in Aeromonas hydrophila water isolates

30 Aeromonas hydrophila water isolates were tested for bacteriocin-like substance (BLS) production using a target panel of closely related microorganisms and other Gram-positive and Gram-negative bacteria, including food-borne pathogens. A. hydrophila showed antibacterial activity against one or more indicator microorganisms, but the activity emerged only with non-phylogenetically related genera or species. In particular all A. hydrophila showed antibacterial activity against one or more of the tested Staphylococcus strains, five against Listeria spp. (Listeria seeligeri, Listeria welshimeri and Listeria ivanovii), and eight presented a weak antagonistic activity towards Streptococcus agalactiae and Lactobacillus spp. Inhibitory activity was not observed against the other Gram-positive (Listeria monocytogenes, Listeria innocua and Enterococcus spp.) and Gram-negative tested strains, including Aeromonas sobria, Aeromonas caviae and the same A. hydrophila, when used as indicator. Anti-staphylococcal activity was observed with a gradual increase of the inhibition zone during incubation and seemed to be influenced by A. hydrophila hemolytic expression. Extrachromosomal analysis showed the presence, in 70% of the strains, of one to five plasmids with molecular masses ranging from 2.1 to 41.5 MDa, but it was not possible to relate this result with BLS production.     

Health status of rudd (Scardinius erythrophthalmus hesperidicus H.) in Lake Vrana on the Island of Cres, Croatia

The health status of rudd ( Scardinius erythrophthalmus hesperidicus H.) in Lake Vrana, the largest Croatian karstic lake, was evaluated. Studies comprising parasitological, haematological and bacteriological surveys were conducted over a 2-year period. Parasitological examination revealed a light infestation of 27% of the examined fish, mostly Dactylogyrus and Ichthyophthirius species. Haematological studies showed that haematocrit values were lower than the physiological limit. A haematocrit coefficient of correlation in all research periods was higher than 15%, indicating that the majority of fish in the study were susceptible to the development of bacterial and other diseases. Indeed, a diverse array of bacteria were isolated from rudd, mainly Flavobacterium spp. and Aeromonas spp., but also some specific fish pathogens, notably Pasteurella piscicida, Yersinia ruckeri , and Edwardsiella ictaluri , were identified. Under stress conditions, detected bacterial species can give rise to disease outbreaks.     

O-Serogrouping and surface components of Aeromonas hydrophila and Aeromonas jandaei pathogenic for eels

Abstract The relationship between virulence, O-serogroup, and some cell-surface features (self-pelleting [SP] and precipitation after boiling [PAB], profile of lipopolysaccharides [LPSs]) and outer membrane proteins [OMPs] was investigated in strains of the pathogenic species Aeromonas hydrophila and A. jandaei isolated from eels. Virulent strains of A. hydrophila reacted mostly with O:19 antiserum, and those of A. jandaei reacted with O:4, O:11, O:15 and O:29 antisera (Guinée and Jansen system). Regarding the PAB and LPS profiles two groups could be distinguished; (i) five PAB⁺ strains of serotype O:19 that possessed a homogeneous O polysaccharide side chain and (ii) thirteen PAB⁻ strains antigenically diverse that either exhibited a heterogenous side chain or were side chain deficient. A major 50 kDa protein was only found in the PAB⁺ strains, whereas major OMPs detected in PAB⁻ strains ranged from 33 to 45 kDa irrespective of the species. Epizootic eel isolates of A. hydrophila belong to serotype O:19 and share cell-surface features with the Aeromonas highly virulent for other hosts. In contrast, epizootic A. jandaei isolates were antigenically diverse. These findings reinforce the importance of an O-serotype as an epidemiological marker in motile Aeromonas strains pathogenic for eels.     

Isolation of Aeromonas hydrophila from cooked tripe

A survey of the microbiological quality of tripe purchased in the Cheshire area was undertaken. Forty-four samples were analysed during a 4 month period in 1991. Total viable counts were frequently high for a cooked product, 40% being 10⁶ or greater. Listeria monocytogenes was isolated by enrichment from 9% of samples. Aeromonas spp. were isolated from 48% of samples. All 11 Aeromonas isolates, sent for further identification, were A. hydrophila. In some instances the count of Aeromonas was very high, being greater than 10⁷ in two samples.     

引起鱼类暴发性流行病的嗜水气单胞菌的血清型、毒力及溶血性

对分离自暴发病鱼的33株嗜水气单胞菌株的血清型、毒力和溶血性进行了研究。结果表明:按O抗原不同,大部分菌株可分为TPS-30和PBJS-76两个血清型,这两个血清型菌株分布于浙江各地及南方其它省市,可能是构成暴发病流行嗜水气单胞的主要血清型;这些菌株对健康白鲫有很强致病力,LD₅₀小于10⁶,有很强的产溶血素能力,但某些菌株的毒力与其溶血效价无直接联系。     

Distribution and survival of motile Aeromonas spp. in brackish water receiving sewage treatment effluent.

The spatiotemporal distributions of Aeromonas spp. and fecal coliforms in a cove receiving sewage treatment effluent and draining into a brackish lagoon were studied for 34 months with sampling at six stations. A total of 452 strains of Aeromonas spp. were isolated and identified at the outflow of the treatment system and at stations in the cove. Hemolytic activity of 289 Aeromonas strains was determined. The Aeromonas spp. and fecal coliform distributions showed seasonal cycles in the pond effluent. These seasonal bacterial cycles were persistent in effluent, at the discharge point, and in the cove. However, the abundance levels of these bacterial distributions decreased gradually from the coast to the open lagoon. A dilution model showed that the Aeromonas spp. and fecal coliform distributions in the cove water were subject not only to dilution effect but also to other environmental factors, such as salinity. A. sobria is the most common species identified in the Aeromonas population present in the cove water. Survival studies confirmed that Aeromonas spp., especially A. sobria, were more sensitive to saline and/or marine stress than fecal coliforms. Among the Aeromonas hydrophila and A. sobria strains, 96 and 97%, respectively, produced hemolysin, whereas among the Aeromonas caviae strains, 95% were nonhemolytic.     

Distribution and survival of motile Aeromonas spp. in brackish water receiving sewage treatment effluent.

The spatiotemporal distributions of Aeromonas spp. and fecal coliforms in a cove receiving sewage treatment effluent and draining into a brackish lagoon were studied for 34 months with sampling at six stations. A total of 452 strains of Aeromonas spp. were isolated and identified at the outflow of the treatment system and at stations in the cove. Hemolytic activity of 289 Aeromonas strains was determined. The Aeromonas spp. and fecal coliform distributions showed seasonal cycles in the pond effluent. These seasonal bacterial cycles were persistent in effluent, at the discharge point, and in the cove. However, the abundance levels of these bacterial distributions decreased gradually from the coast to the open lagoon. A dilution model showed that the Aeromonas spp. and fecal coliform distributions in the cove water were subject not only to dilution effect but also to other environmental factors, such as salinity. A. sobria is the most common species identified in the Aeromonas population present in the cove water. Survival studies confirmed that Aeromonas spp., especially A. sobria, were more sensitive to saline and/or marine stress than fecal coliforms. Among the Aeromonas hydrophila and A. sobria strains, 96 and 97%, respectively, produced hemolysin, whereas among the Aeromonas caviae strains, 95% were nonhemolytic.     

草鱼的一种急性细菌性传染病病原的分离鉴定及致病性研究

草鱼(Ctenopharyngodon idellus)的细菌性疾病是草鱼人工养殖过程中经常出现的疾病。文章对河南一草鱼养殖场2009年8月出现的暴发性细菌性传染病进行了研究。该渔场饲养的草鱼成鱼和鱼苗出现了体表溃烂、局部出血, 肠系膜充血、出血, 腹腔积水等症状, 并大量死亡。从濒死草鱼的内脏中分离到3株细菌, 经形态学、生理生化特性及16S rDNA序列分析, 鉴定为霍乱弧菌(Vibrio cholerae)、嗜水气单胞菌(Aeromonas hydrophila)和柱状黄杆菌(Flavobacterium columnare)。其中, 从患病鱼苗体内分离到霍乱弧菌; 从成鱼分离到嗜水气单胞菌和柱状黄杆菌。回归试验证明3株细菌均能使草鱼致死, 其中霍乱弧菌和嗜水气单胞菌对草鱼的半数致死量(LD50)分别是0.15104 cfu/g和0.96103 cfu/g。从草鱼中分离到致病性霍乱弧菌是首次报道。药敏试验表明, 在所试的17种抗生素中, 霍乱弧菌仅对利福平、左旋氧氟沙星、链霉素、奥复星、庆大霉素等5种抗生素敏感; 嗜水气单胞菌仅对菌必治、奥复星、庆大霉素等3种抗生素敏感; 而柱状黄杆菌则对其中的氨苄青霉素、利福平、左旋氧氟沙星等10种抗生素均敏感。       

Epidemiology and potential land-sea transfer of enteric bacteria from terrestrial to marine species in the Monterey Bay Region of California

Marine mammals are at risk for infection by fecal-associated zoonotic pathogens when they swim and feed in polluted nearshore marine waters. Because of their tendency to consume 25-30% of their body weight per day in coastal filter-feeding invertebrates, southern sea otters (Enhydra lutris nereis) can act as sentinels of marine ecosystem health in California. Feces from domestic and wildlife species were tested to determine prevalence, potential virulence, and diversity of selected opportunistic enteric bacterial pathogens in the Monterey Bay region. We hypothesized that if sea otters are sentinels of coastal health, and fecal pollution flows from land to sea, then sea otters and terrestrial animals might share the same enteric bacterial species and strains. Twenty-eight percent of fecal samples tested during 2007-2010 were positive for one or more potential pathogens. Campylobacter spp. were isolated most frequently, with an overall prevalence of 11%, followed by Vibrio cholerae (9%), Salmonella spp. (6%), V. parahaemolyticus (5%), and V. alginolyticus (3%). Sea otters were found positive for all target bacteria, exhibiting similar prevalences for Campylobacter and Salmonella spp. but greater prevalences for Vibrio spp. when compared to terrestrial animals. Fifteen Salmonella serotypes were detected, 11 of which were isolated from opossums. This is the first report of sea otter infection by S. enterica Heidelberg, a serotype also associated with human clinical disease. Similar strains of S. enterica Typhimurium were identified in otters, opossums, and gulls, suggesting the possibility of land-sea transfer of enteric bacterial pathogens from terrestrial sources to sea otters.     

Distribution of Aeromonas species in waters with different levels of pollution

Strains of Aeromonas spp. (883) were isolated from 10 stations in the north-west of Spain. Biotyping of the strains gave: 55% Aeromonas caviae, 34% A. hydrophila, 6% A. sobria and 5% Aeromonas spp. Phenotypic characters that have been claimed to be related to virulence such as haemolysis and the Voges-Proskauer reaction were detected mostly in A. hydrophila and A. sobria. The distribution of the species was significantly related to levels of faecal pollution in waters. Aeromonas caviae predominated in sewage and waters with a high degree of faecal pollution. In less polluted waters, either fresh or marine, A. caviae and A. hydrophila were almost equally distributed. In waters with low or no faecal pollution, the proportion of A. sobria to other species increased considerably.     

Distribution of Aeromonas species in waters with different levels of pollution

Strains of Aeromonas spp. (883) were isolated from 10 stations in the north-west of Spain. Biotyping of the strains gave: 55% Aeromonas caviae , 34% A. hydrophila , 6% A. sobria and 5% Aeromonas spp. Phenotypic characters that have been claimed to be related to virulence such as haemolysis and the Voges-Proskauer reaction were detected mostly in A. hydrophila and A. sobria. The distribution of the species was significantly related to levels of faecal pollution in waters. Aeromonas caviae predominated in sewage and waters with a high degree of faecal pollution. In less polluted waters, either fresh or marine, A. caviae and A. hydrophila were almost equally distributed. In waters with low or no faecal pollution, the proportion of A. sobria to other species increased considerably.     

Pathogenicity of live bacteria and extracellular products of motile Aeromonas isolated from eels

The pathogenic activities in vitro and in vivo of live bacteria and extracellular products (ECP) of 24 motile Aeromonas strains were investigated. Most Aer. hydrophila and Aer. jandaei isolates were pathogenic for eels (LD₅₀ 10^5·4-10^7·6 cfu fish^-1) but no Aer. sobria , Aer. caviae and Aer. allosaccharophila caused mortality in eels at doses of > 10^8·4 cfu fish^-1. Of these Aeromonas strains, Aer. hydrophila and Aer. jandaei in particular produced elastases and haemolysins against fish erythrocytes. ECP from Aer. hydrophila and Aer. jandaei caused degenerative changes in fish cell lines and were strongly toxic for eels (LD⁵⁰ 1·0–3·2 μg (g fish)^-1) reproducing the symptoms associated with natural disease. ECP from non-pathogenic species were inactive on fish cell lines as well as being poorly lethal for eels (LD⁵⁰ > 9·2 μg (g fish)^-1). All these biological activities of Aeromonas ECP were lost after heat treatment. These findings indicate differences between pathogenic and non-pathogenic Aeromonas species with respect to the expression of virulence factors, and show that elastases, haemolysins and exotoxins play a leading role in the pathogenicity of motile Aeromonas for eels.     

An enzyme-linked immunosorbent assay for detection of Vibrio vulnificus biotype 2: development and field studies.

Vibrio vulnificus biotype 2 is a primary eel pathogen which constitutes a lipopolysaccharide (LPS)-based homogeneous O serogroup within the species. In the present work, we have developed an enzyme-linked immunosorbent assay (ELISA) based on the specificity of LPS for the detection of this pathogen. The ELISA specificity was confirmed after testing 36 biotype 2 strains from laboratory cultures and environmental samples, 31 clinical and environmental biotype 1 isolates, and several strains of Vibrio, Aeromonas, and Yersinia species, including the fish pathogens V. anguillarum, V. furnissii, A. hydrophila, and Y. ruckerii. The detection limits for biotype 2 cells were around 10(4) to 10(5) cells/well, and the immunoassay was also able to detect cells in the nonculturable state. Artificially infected eels and environmental samples were analyzed, and the immunodetection was confirmed by cultural methods (isolation on selective and nonselective media before and after broth enrichment). With this methodology, V. vulnificus biotype 2 was successfully detected in infected eels and asymptomatic carriers, which suggests that eels can act as a reservoir for this pathogen.     

Identification of Aeromonas hydrophila hybridization group 1 by PCR assays.

Synthetic oligonucleotide primers of 24 and 23 bases were used in a PCR assay to amplify a sequence of the lip gene, which encodes a thermostable extracellular lipase of Aeromonas hydrophila. A DNA fragment of approximately 760 bp was amplified from both sources, i.e., lysed A. hydrophila cells and isolated DNA. The amplified sequence was detected in ethidium bromide-stained agarose gels or by Southern blot analysis with an internal HindIII-BamHI 356-bp fragment as a hybridization probe. With A. hydrophila cells, the sensitivity of the PCR assay was < 10 CFU, and with the isolated target, the lower detection limit was 0.89 pg of DNA. Primer specificity for A. hydrophila was determined by the PCR assay with cells of 50 strains of bacteria, including most of the 14 currently recognized DNA hybridization groups of Aeromonas spp. as well as other human and environmental Aeromonas isolates. Detection of A. hydrophila by PCR amplification of DNA has great potential for rapid identification of this bacterium because it has proved to be highly specific.     

Aeromonas hydrophila: Ecology and Toxigenicity of Isolates from an Estuary

A microbiological survey of Aeromonas hydrophila in Chesapeake Bay and its tributaries showed that this species is ubiquitous, occurring in numbers ranging from <0.3/l to 5 × 10³/ml in the water column and ca. 4.6 × 10²/g in sediment. It was recovered from water samples collected at several locations in Chesapeake Bay representing various salinity regimes, but the numbers of A. hydrophila in higher salinity water, i.e. 15^O/OO, were low. Results of stepwise multiple linear regression analysis showed that concentrations of A. hydrophila were correlated with total, aerobic, viable, heterotrophic, bacterial counts, and, in addition, were inversely related to salinity and to concentration of dissolved oxygen. Seasonal occurrence was recorded, with fewer strains of A. hydrophila encountered during the winter months. The potential pathogenicity of A. hydrophila strains isolated from Chesapeake Bay was estimated by testing selected isolates for toxigenicity, using the Y-1 adrenal cell assay. Of 116 isolates tested, 83 (71%) produced a cytotoxic response, a characteristic found to be correlated with the lysine decarboxylase and Voges-Proskauer reactions. Eight of 11 strains tested, which elicited fluid accumulation in the rabbit ligated ileal loop assay, also provoked a cytotoxic reaction in the Y-l adrenal cell assay. Results of the study indicate that large numbers of toxigenic A. hydrophila can be found in an estuary and such strains may be pathogenic for man and/or animals.     

Occurrence of Staphylococcus and enteropathogens in soft cheese commercialized in the city of Rio de Janeiro,Brazil

AIMS: To investigate the presence of Staphylococcus aureus, enteropathogenic Escherichia coli (EPEC), Aeromonas spp. and Yersinia spp. in soft cheese commercialized in Rio de Janeiro, Brazil. METHODS AND RESULTS: A total of 45 samples of cheese from three different brands marketed in Rio de Janeiro city were analysed for faecal coliform levels using the Most Probable Number (MPN) technique. The samples were also analysed using conventional methodology for the investigation of food-borne pathogens.High levels of faecal contamination were detected in 95.5% of cheese samples. Staphylococcus aureus was isolated from 20% of samples, of which 17.7% were above the limits allowed by Brazilian legislation. Aeromonas hydrophila and Aer. caviae were detected in 17.7% of the samples. Yersinia spp. were not found in this study. EPEC was isolated from 21.1% of the samples and the most frequently found serogroups were O127, followed by O55 and O26. CONCLUSIONS: Our results showed that 95.5% of cheese samples had high levels of faecal coliforms. The isolation of Staph. aureus, serogroups of EPEC and Aeromonas spp. suggested that the soft cheese commercialized in the city of Rio de Janeiro may represent a health risk for the consumers. SIGNIFICANCE AND IMPACT OF THE STUDY: These results suggest that soft cheese may act as an important vehicle of transmission for well-established pathogens.     

Contaminated marine wounds--the risk of acquiring acute bacterial infection from marine recreational beaches.

An animal model was used to determine the potential for causing wound infections of bacteria isolated from marine recreational beaches in Hong Kong. Water samples were characterized physically, chemically and bacteriologically and used to inoculate artificially-induced wounds in rats. Morbidity and mortality correlated significantly (P < 0.01) with MacConkey plate counts and faecal coliform counts (membrane filtration) and inversely with salinity of the water. The majority of deaths were due to infection caused by marine and estuarine bacteria rather then enteric organisms. A total of 318 bacterial strains was isolated from the wounds and blood of animals inoculated with seawater, of which 242 were marine/estuarine (predominantly Vibrio spp., Aeromonas hydrophila and Pseudomonas putrefaciens) and 40 were enterobacteria. The virulence of the animal strains were comparable with those from clinical sources.