Chemotaxis ofAeromonas hydrophila to the surface mucus of fish

Isolates ofAeromonas hydrophila from various sources show different chemotactic responses to mucus from the surface of freshwater fish. Some isolates were nonchemotactic to fish surface mucus. Isolates ofA. hydrophila from fish lesions had a significantly higher chemotactic index than isolates ofA. hydrophila from water. Maximum chemotactic responses occurred more often to diluted fish mucus than to undiluted samples. Fish which were experimentally stressed did not produce mucus that was more or less chemotactic than that of unstressed fish. Fish with red-sore lesions produced surface mucus which was not chemotactic toA. hydrophila. Differences between fish, for any isolate, were also not significant. The chemotactic substance(s) in fish mucus has a molecular weight of approximately 100,000 and did not appear to be labile when heated to 56°C.     

Ecology ofAeromonas hydrophila in a South Carolina cooling reservoir

Densities ofAeromonas hydrophila were determined monthly from December 1975 to December 1977 in a South Carolina cooling reservoir which receives heated effluent from a single nuclear production reactor. Selected water quality parameters and prevalence of red-sore disease among largemouth bass were monitored simultaneously.Higher densities ofA. hydrophila were observed in areas of the reservoir receiving effluent from the reactor. Densities ofA. hydrophila generally were heterogeneous in the water column. The sediments had lower densities ofA. hydrophila than water immediately above.A. hydrophila could not be isolated from sediments greater than 1 cm from the water interface. Temperature, redox potential, pH, and conductivity were all significantly correlated with densities ofA. hydrophila in the water column. The temporal and spatial distribution and abundance ofA. hydrophila in water were not related to total organic carbon, dissolved organic carbon, particulate organic carbon, inorganic carbon, or dissolved oxygen. High densities ofA. hydrophila were observed in mats of decomposingMyriophyllum spicatum and, enterically, in largemouth bass, several other species of fish, turtles, alligators, and snails. The greatest densities ofA. hydrophila in water occurred during March and June with a second peak in October. The mean monthly densities ofA. hydrophila were positively correlated with the incidence of infection in largemouth bass. Largemouth bass from thermally altered parts of the reservoir had a significantly higher incidence of infection. It is concluded that thermal effluent significantly affects the ecology ofA. hydrophila and the epizootiology of red-sore disease within Par Pond.     

The protozoan epizooites found on the gills of Gammarus pulex

Four protozoan epizooites are found attached to the gills of Gammarus pulex collected from Rutland Water. The suctorian, Dendrocometes paradoxus, and the peritrichs, Epistylis sp. and Lagenophrys sp., are relatively abundant, but the chonotrich, Spirochona gemmipara is only occasionally found. SEM studies of various aspects of these protozoa are described. SEM has been useful in distinguishing between the two species of Lagenophrys found on Gammarus. L. ampulla is found on the gills and L. nassa is found on the limbs. The two species are distinguished by the differences in the structure of the loricastome. The relationship between the epizooites and their host is discussed briefly.     

Description of a New Freshwater Ciliate Epistylis wuhanensis n. sp. (Ciliophora,Peritrichia) from China,with a Focus on Phylogenetic Relationships within Family Epistylididae

Two populations of Epistylis wuhanensis n. sp., a new freshwater peritrich ciliate, were isolated from different freshwater ponds located in Hubei, China. Their morphological characteristics were investigated using live observation, protargol impregnation, and scanning electron microscopy (SEM). Specimens from the two populations showed identical arrangement of the infraciliature and identical small subunit ribosomal RNA (SSU rRNA) gene and ITS1‐5.8S‐ITS2 sequences. The zooids present bell‐shaped and 90–175 × 27–54 μm in vivo. Macronucleus is variable in shape and located in the middle of cell. Pellicle is usually smooth with 139–154 and 97–105 striations above and below the trochal band, respectively. SSU rRNA gene and ITS1‐5.8S‐ITS2 sequences of E. wuhanensis n. sp. did not match any available sequences in GenBank. Phylogenetically, E. wuhanensis n. sp. clusters with the other Epistylis within the family Epistylididae, but is distinct from the major clades of Epistylis. Above all, the morphological characteristics and molecular analyses support that the present Epistylis is a new species. Expanded phylogenetic analyses of sessilids based on both SSU rRNA gene sequences and ITS1‐5.8S‐ITS2 sequences reveal that the genus Epistylis consists of Epistylis morphospecies and taxonomic revision of the genus is needed.     

Ultrastructural Features of Three Ectocommensal Protozoa Attached to the Gills of the Red Swamp Crawfish,Procambarus clarkii (Crustacea: Decapoda)1

Because heavy branchial infestations are thought to interfere with respiration, we examined the attachment of three stalked ciliates commonly found in the branchial chambers of Louisiana crawfish. Attachments by Cothurnia sp., Epistylis sp. and Acineta sp. differ in their fine structure. Stalks of the peritrichous ciliates. Cothurnia and Epistylis, contain striated tubules that differ in their arrangement, diameter, and in the periodicity of their striations. In both species the striated tubules branch within the basal disk and attach to a pad of adhesive material secreted by the organism during initial attachment to the gill surface. The stalk of the suctorian Acineta is composed of a striated honeycomb-like matrix. Within the basal disc the matrix is disorganized; however, striated elements anchor the stalk to a pad of adhesive material. Attachment sites also differ in the amount of secretory material deposited. Cothurnia forms a multi-layered, granular pad; Epistylis forms an indistinct, microfibrillar layer, and Acineta deposits a thick mucoid pad. None of the ciliates appear to damage the gill epicuticle nor is there an obvious host response. Harmful effects are probably limited to a decrease in respiratory surface area and disruption of normal water flow patterns. This may impair respiration sufficiently to increase the susceptibility of crawfish to low dissolved oxygen concentrations encountered periodically in commercial crawfish ponds.     

Development of diagnostic and vaccine markers through cloning, expression, and regulation of putative virulence-protein-encoding genes of Aeromonas hydrophila

Aeromonas hydrophila is an opportunistic bacterial pathogen that is associated with a number of diseases in fish, amphibians, reptiles, and humans. In fish it causes several disease symptoms including tail and skin rot, and haemorrhagic septicemia; in human it causes soft-tissue wound infection and diarrhoea. The pathogenesis of A. hydrophila is multifactorial, but the mechanism is unknown so far. It is considered to be mediated by expression and secretion of extracellular proteins such as aerolysin, lipase, chitinase, amylase, gelatinase, hemolysins, and enterotoxins. A number of the putative virulence-protein-encoding genes that are present in the genome of A. hydrophila have been targeted by PCR for molecular diagnosis. These significant genes are also targeted for over-production of proteins by cloning and expression methods. In this review, we emphasize recent progress in the cloning, expression, and regulation of putative virulence-protein-encoding genes of A. hydrophila for a better understanding of the pathogenesis and also help to provide effective strategies for control of diseases.     

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.     

Cytopathology, Mode of Aphid Transmission and Search for the Casual Agent of Cotton Bunchy Top Disease

The cytopathological effects of cotton bunchy top (CBT) disease and its mode of transmission by Aphis gossypii Glover (cotton aphid), were studied. CBT infection affected the leaf epidermal layer producing a loose, ruptured and rough surface morphology with many stomata closed and misshapen. Roots of CBT‐infected plants showed reduced growth, small knots and a dark brown appearance. A single aphid per plant was capable of transmitting CBT at 5%, whereas three aphids per plant transmitted CBT to 50% of the cotton seedlings and 20 aphids per plant transmitted the disease agent to 80% of the cotton seedlings. Aphis gossypii acquired CBT after a minimum acquisition access period of 5 min and transmitted the agent after a minimum inoculation access period of 1 h. Both alate and apterous aphids and nymph instars 2, 3 and 4 of A. gossypii transmitted CBT. This preliminary data suggest that A. gossypii transmits CBT in a semi‐persistent manner. Myzus persicae Sulz (green peach aphid) was unable to transmit CBT. A comprehensive attempt to isolate the CBT agent, using a range of virological techniques including double‐stranded RNA extraction, two‐dimensional gel electrophoresis for viroid, circular DNA test, nanovirus polymerase chain reaction (PCR), luteovirus PCR and enzyme‐linked immunosorbent assay, phytoplasma test, nucleoprotein purification and electron microscopy, was unsuccessful, raising the possibility that CBT may be caused by a unique new pathogen.     

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.     

Relationship of season, thermal loading and red-sore disease with various haematological parameters in Micropterus salmoides

One hundred and fifty largemouth bass (Micropterus salmoides) were caught during the mid-winter and mid-summer months in Par Pond, an 1120 ha cooling reservoir located on Savannah River Plant near Aiken, SC. Equal numbers of fish were taken from ambient and thermally altered (> 10° C ambient) areas of the lake. The total length and weight of each bass was noted, along with the presence or absence of lesions associated with red-sore disease (caused by the bacterium, Aeromonas hydrophila and the ciliate, Epistylis sp.). Blood was drawn, by heart puncture, from each bass and the following haematological parameters measured: haematocrit, haemoglobin, iron, total iron-binding capacity, thyroxine, sodium and potassium. Only the total iron-binding capacity was significantly higher in bass with red-sore disease; none of the other blood parameters were significantly different in infected and uninfected bass. Bass from thermally altered locations had consistently higher haematocrits during both summer and winter. Bass from thermal and ambient locations were not different with respect to iron, iron-binding capacity, sodium or potassium levels. However, bass taken during summer months had higher levels of circulating iron than individuals in winter months. Summer bass had higher thyroxine levels than bass in winter. These results are discussed in terms of their relationship to red-sore disease, season and the impact of thermal effluent.     

Dietary Administration of Probiotic Aeromonas veronii V03 on the Modulation of Innate Immunity,Expression of Immune-Related Genes and Disease Resistance Against Aeromonas hydrophila Infection in Common Carp (Cyprinus carpio)

This study investigates the effects of dietary Aeromonas veronii V03 supplementation on growth performances, innate immunity, and expression of immune-related genes in lymphoid organs of Cyprinus carpio and resistance to Aeromonas hydrophila infection. Fish were fed for 4 weeks with basal diet (BD; without probiotic), and experiment diet containing different doses of A. veronii V03 at 3.2 × 10⁷ (DI) and 3.5 × 10⁹ (DII) CFU g⁻¹ of diet. At the end of the probiotic feeding trial, fish were challenged with A. hydrophila, and the percentage of survival rates was recorded over 7 days. Results revealed that fish fed with A. veronii V03 demonstrated a significant improvement in growth and enhancement of innate immunity, including respiratory burst, myeloperoxidase, and lysozyme activities, and total immunoglobulin level compared with BD fed to fish. Relatively, expression of cytokines (MyD88, IL-1β1, IL-8, and IL-10) and c- and g-type lysozymes were significantly up- and downregulated in lymphoid organs of fish. Moreover, dietary supplementation of A. veronii V03 exhibited significantly (p < 0.001) higher survival rates of DI (90%) and DII (96.66%) compared with BD (53.33%) fed fish against A. hydrophila infection. These findings help to understand the effects of probiotic A. veronii V03 administrated feed influences on growth and ailment resistance to A. hydrophila infection by regulating innate and systemic immunity in common carp fish.

     

Changes in adhesion ability of Aeromonas hydrophila during long exposure to salt stress conditions

Aims

Stressful environmental conditions influence both bacterial growth and expression of virulence factors. In the present study, we evaluated the influence of NaCl on Aeromonas hydrophila adhesiveness at two temperatures. This agent is often involved in clinical cases; however, its pathogenic potential is still not fully understood.

Methods and Results

Bacteria were grown in presence of 1·7%, 3·4%, 6·0% NaCl over a 188 day period and then reinoculated in fresh Nutrient Broth with incubation at 4 and 24°C. Bacterial adhesiveness was tested on Hep‐2 cells, and specimens were processed for light, scanning and transmission electron microscopy. Adhesive capacity decreased over time with an increase in reduction percentages depending on NaCl concentrations. At 1·7% NaCl, the reduction was apparently temporary and adhesiveness rapidly recovered in revitalized bacteria, while 3·4%, 6·0% NaCl seemed to be detrimental. Normal, elongated and filamentous bacteria retained adhesiveness capability, although with reduced expression, while in spherical cells, this property seemed to be lost or dramatically reduced.

Conclusions

Our study shows that high osmolarity plays a significant role in adhesion inhibition, therefore having possible implications in the pathogenesis of the infections by Aer. hydrophila.

Significance and Impact of the Study

This study intends to give a contribution to a better understanding of the pathogenic role of this bacterium whose pathogenicity is still under debate.     

Isolation and Identification of a Novel Inducible Antibacterial Peptide from the Skin Mucus of Japanese Eel, <Emphasis Type="Italic">Anguilla japonica</Emphasis>

In this study, acetone extracts and acidic extracts were prepared from skin mucus, gill, kidney, liver and spleen of the Japanese eel, Anguilla japonica, and they exhibited different levels of antibacterial activities against three strains of Gram-negative bacteria, Edwardsiella tarda, Aeromonas hydrophila, Aeromonas sp. and one Gram-positive bacterium Micrococcus leteus. The mucus was chosen as the source of antibacterial peptide for further purification of antibacterial peptides. Following the intraperitoneal injection of A. hydrophila, one of the main pathogenic bacteria of Japanese eel and many other fish, a peptide was purified from acetic acid extraction of the skin mucus, by using cationic exchange liquid chromatography and reverse-phase high-performance liquid chromatography (RP-HPLC). The isolated antibacterial peptide, named as AJN-10, exhibited antibacterial activity against A. hydrophila. The AJN-10 is a heat-tolerant and hydrophilic peptide. The molecular weight of this peptide is 6,044.28 Da, as determined by matrix-assisted laser desorption ionisation time of flight mass spectrometry. The 20 N-terminal amino acid sequences were clarified by Edman degradation, and based on results of homology search by BLAST analysis of the 20 N-terminal sequences, the AJN-10 showed little similarity to other proteins in databases.     

Neutrophil activity affects Oreochromis mossambicus (Peters, 1852) antibody production against heat‐killed Aeromonas hydrophila vaccine

Aeromonas hydrophila is one of the important and most common pathogens of warm water fish. Prophylactic and therapeutic measures against A. hydrophila infection are essential to prevent loss of fish production in aquaculture. A heat‐killed vaccine was developed against three strains of A. hydrophila, namely O21, O26 and O28, and analysed for their comparative immunogenicity in Oreochromis mossambicus (Peters, 1852). Studied were the neutrophil activity and specific antibody response of the host against the vaccines, which showed that neutrophil activity was highest for the heat‐killed O21, but that the heat‐killed O28 produced the highest antibody titres. The antibody cross‐reactivity tests indicated that the antibody raised against O28 was pan‐reactive whereas it was less cross‐reactive in O21. Thus strain O28 may be used as a vaccine candidate for a pan‐protection of fish from various strains of A. hydrophila infections. However, further rigorous studies with different fish species and bacterial strains are needed to confirm these results.     

Developmental Regulation of Susceptibility and Tolerance to Fusarium Root Rot in Beans

The soil-borne fungus, Fusarium solani f. sp. phaseoli, attacks roots and hypocotyls of bean (Phaseolus vulgaris) plants causing a devastating disease called root and foot rot. In a study of the host-pathogen relationship it was found that young bean roots, with the radicle just emerging, were highly tolerant to the pathogen, whereas older bean seedlings, with a fully developed root system, were completely susceptible. Investigations by low-temperature scanning electron microscopy demonstrated that significantly fewer spores and hyphae were present on the root surface of young bean seedlings as compared to older ones. A similar pattern of attachment was found when bean roots were inoculated with spores of F. solani f. sp. pisi, a related pathogen causing disease on peas but not on beans. Light microscopic studies showed that F. solani f. sp. pisi did not penetrate the root but rapidly formed thick-walled resting spores on the root surface. F. solani f. sp. phaseoli on the other hand quickly penetrated the root and formed an extensive network of fungal hyphae. These results demonstrate that the ability of fungal propagules to adhere to and to penetrate host tissues are two distinct processes. Furthermore, the data indicate that young bean roots lack a surface component necessary for attachment of fungal spores which may help explain their tolerance to Fusarium root rot.     

Tumors in fish from the Detroit River

A fish tumor survey was conducted in the Lower Detroit River during 1985 to 1987. Five species of fish were collected from sediment deposition zones and were examined for neoplasia. Neoplasms and related lesions were found in bullhead (Ictalurus nebulosus), walleye (Stizostedion vitreum), redhorse sucker (Moxostoma, sp), white sucker (Catostomus commersoni), and bowfin (Amia calva). Overall, 8.2 percent of the fish examined had oral/dermal lesions and 10.1 percent had liver lesions. Liver and skin tumors were found to be age-related in bullheads and size-related in walleye. Based on the fish surveyed, the incidence of tumors in fish from the Detroit River was similar to that observed in other chemically contaminated waterways.     

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.     

The effect of Euglena viridis on immune response of rohu, Labeo rohita (Ham.)

The study evaluated the effect of dietary doses of Euglena viridis on the immune response and disease resistance of Labeo rohita fingerlings against infection with the bacterial pathogen Aeromonas hydrophila. L. rohita fingerlings were fed with diet containing 0 (Control), 0.1 g, 0.5 g, 1.0 g Euglena powder kg⁻¹ dry diet for 90 days. Biochemical (serum total protein, albumin, globulin, albumin:globulin ratio), haematological (WBC, RBC, haemoglobin content) and immunological (superoxide anion production, lysozyme, serum bactericidal activity) parameters of fish were examined after 30, 60 and 90 days of feeding. Fish were challenged with A. hydrophila 90 days post-feeding and mortalities were recorded over 10 days post-infection. The results demonstrate that fish fed with Euglena showed increased levels of superoxide anion production, lysozyme, serum bactericidal activity, serum protein and albumin (P < 0.05) compared with the control group. Following challenge with A. hydrophila less survivability was observed in the control group (56.65%) than the group fed the experimental diets. The group fed 0.5 g Euglena kg⁻¹ dry diet showed the highest percentage survival (75%). These results indicate that Euglena stimulates the immunity and makes L. rohita more resistant to A. hydrophila infection.     

On the morphology of antennular sensory and attachment organs in cypris larvae of the deep‐sea vent/seep barnacles,Ashinkailepas and Neoverruca

Barnacle cypris larvae show high morphological variation in the organs used in search of and attaching to a substratum. This variation may represent adaptation to the habitat of the species. Here, we studied SEM level morphologies of cypris antennular sensory and attachment organs in a deep‐sea vent endemic species (Neoverruca sp.) and a vent/seep inhabiting species (Ashinkailepas seepiophila). We compare them with three species from other environments. The antennular morphologies of Neoverruca sp. and A. seepiophila were similar, which is consistent with recent molecular studies showing a close relationship of the two species. The setation pattern of the antennules was very conservative among species from various environments. In contrast, striking differences were observed in the structure of the attachment organ (the third antennular segment). Neoverruca sp. and A. seepiophila had no velum or a skirt surrounding the attachment disc on the third segment, while other cirripede cyprids almost always have either of these structures. In addition, both cyprids of A. seepiophila and Neoverruca sp. had the attachment disc angled toward the substratum, whereas it faces distally in cyprids from hard bottom inhabiting barnacles. We suggest that both velum/skirt and the angle of the attachment disc play an important role, when the antennules are contacting the substratum during surface exploration. Differences in attachment organ structures may be highly adaptive, enabling cirripede species to enter new habitats during evolution. J. Morphol. 277:594–602, 2016. © 2016 Wiley Periodicals, Inc.