Vibrio carchariae,a pathogen of the abalone Haliotis tuberculata

Since 1997, mass mortality of the abalone Haliotis tuberculata L. has occurred in the natural environment along the French coast. The outbreak of disease started on the south coast of Brittany near Concarneau in 1997, then spread to the north of Brittany (in 1998) and the west coast of Normandy (Golfe de St. Malo in 1999). Between 60 and 80% of the abalone died. In 1999, mortality also affected a land-based abalone farm in Normandy during the summer. At this farm, a Vibrio sp. was isolated in abundance from abalone that had just died. The disease was experimentally reproduced by inoculation or by introducing the pathogen into the surrounding water. This vibrio, identified by genotypic and phenotypic characters, is related to V carchariae. It is similar to the V carchariae, responsible for mortality in the Japanese abalone Sulculus diversicolor supratexta, but some phenotypic characters differentiate both strains. In 2000, healthy abalone placed in 2 sites on the north and south coasts of Brittany died, and the pathogen V carchariae could be isolated from dead individuals, demonstrating that the pathogen was probably the cause of the abalone disease that has been occurring since 1997 in Brittany.     

Protective Efficacy of a Pseudoalteromonas Strain in European Abalone,Haliotis tuberculata,Infected with Vibrio harveyi ORM4

Probiotics and Antimicrobial Proteins - The hemolymph of healthy marine invertebrates is known to harbor antibiotic-producing bacteria belonging to the genus Pseudoalteromonas. Such strains are...     

Vibrio harveyi Adheres to and Penetrates Tissues of the European Abalone Haliotis tuberculata within the First Hours of Contact

Vibrio harveyi is a marine bacterial pathogen responsible for episodic epidemics generally associated with massive mortalities in many marine organisms, including the European abalone Haliotis tuberculata. The aim of this study was to identify the portal of entry and the dynamics of infection of V. harveyi in the European abalone. The results indicate that the duration of contact between V. harveyi and the European abalone influences the mortality rate and precocity. Immediately after contact, the epithelial and mucosal area situated between the gills and the hypobranchial gland was colonized by V. harveyi. Real-time PCR analyses and culture quantification of a green fluorescent protein-tagged strain of V. harveyi in abalone tissues revealed a high density of bacteria adhering to and then penetrating the whole gill-hypobranchial gland tissue after 1 h of contact. V. harveyi was also detected in the hemolymph of a significant number of European abalones after 3 h of contact. In conclusion, this article shows that a TaqMan real-time PCR assay is a powerful and useful technique for the detection of a marine pathogen such as V. harveyi in mollusk tissue and for the study of its infection dynamics. Thus, we have revealed that the adhesion and then the penetration of V. harveyi in European abalone organs begin in the first hours of contact. We also hypothesize that the portal of entry of V. harveyi in the European abalone is the area situated between the gills and the hypobranchial gland.     

Effect of nitrite on immune response of Taiwan abalone Haliotis diversicolor supertexta and its susceptibility to Vibrio parahaemolyticus

Taiwan abalones Haliotis diversicolor supertexta held in 30% per thousand seawater and 26 degrees C were injected with tryptic soy broth (TSB)-grown Vibrioparahaemolyticus (1.6 x 10(5) CFU [colony-forming units] abalone(-1)), and then placed in water containing different concentrations of nitrite-N (nitrite as nitrogen): 0.01 mg l(-1) (control), 1.05, 3.04, 5.10 and 10.06 mg l(-1). Mortality of the abalones increased in direct parallel to ambient nitrite-N concentration. Over 12 to 48 h, the mortality of V. parahaemolyticus-injected abalones held in 3.04 mg l(-1) nitrite-N was significantly higher than that of abalones in the control solution. Abalones that had been exposed to control, 0.96, 2.95, 5.03 and 10.16 mg l(-1) nitrite-N for 24, 72 and 120 h were examined for THC (total hemocyte count), phenoloxidase activity, respiratory bursts (release of superoxide anion), phagocytic activity, and clearance efficiency of V. parahaemolyticus. The THC increased in abalone after 72 h exposure to 0.96 and 2.95 mg l(-1) nitrite-N, but decreased in abalones after 24 h exposure to 5.03 and 10.16 mg l(-1) nitrite-N. Phenoloxidase activity and respiratory bursts increased, while phagocytic activity and clearance efficiency decreased in abalones exposed to > or = 0.96 mg l(-1) nitrite-N for 24 h. It is concluded that nitrite-N in water at concentrations as low as 0.96 mg l(-1) weakens the immune response and increases mortality of H. diversicolor supertexta infected with V. parahaemolyticus.     

Effect of ammonia on the immune response of Taiwan abalone Haliotis diversicolor supertexta and its susceptibility to Vibrio parahaemolyticus

Taiwan abalone Haliotis diversicolor supertexta held in 30 parts/per thousand seawater and 26 degrees C were injected with TSB-grown Vibrio parahaemolyticus (1.6 x 10(5)cfu abalone(-1)), and then placed in water containing different concentrations of ammonia-N (un-ionized plus ionized ammonia) at 0.01 mg l(-1) (control), 1.12, 3.22, 5.24 and 10.18 mg l(-1). Mortality of abalone increased directly with ambient ammonia-N concentration. After 12 h, the mortality of V. parahaemolyticus-injected abalone held in 3.22 mg l(-1) ammonia-N was significantly higher than those placed in 1.12 mg l(-1) ammonia-N and the control solution. In another experiment, the abalone which had been exposed to control, 1.08, 3.16, 5.37 and 10.34 mg l(-1) ammonia-N for 24, 72 and 120 h were examined for THC (total haemocyte count), phenoloxidase activity, respiratory burst (release of superoxide anion), and phagocytic activity and clearance efficiency to V. parahaemolyticus. The abalone when exposed to 3.16 mg l(-1) ammonia-N had decreased THC after 72 h, and decreased phenoloxidase activity, phagocytic activity and clearance efficiency after 24 h. However, the abalone when exposed to 3.16 mg l(-1) ammonia-N had increased respiratory burst after 24 h. The immune parameters except superoxide anion seemed to be suppressed in a dose-dependent fashion after 24 h. It is concluded that ammonia caused a depression in immune parameters and an increase in mortality of H. diversicolor supertexta from V. parahaemolyticus infection.     

Change in water temperature on the immune response of Taiwan abalone Haliotis diversicolor supertexta and its susceptibility to Vibrio parahaemolyticus

Taiwan abalones, Haliotis diversicolor supertexta, held in 30 parts/per thousand seawater at 28 degrees C, were injected with TSB-grown Vibrio parahaemolyticus (1.6x10(5) cfu abalone(-1)) and then transferred to 20, 24, 28 and 32 degrees C. All abalones transferred to 32 degrees C died by 72 h. The mortality of V. parahaemolyticus-injected abalone held at 20 and 24 degrees C was significantly lower over 24-96 h, compared to animals held at 28 and 32 degrees C. In a separate experiment designed to measure immune function, abalones held in 30 per thousand seawater at 28 degrees C and then transferred to 20, 24, 28 and 32 degrees C were examined for total haemocyte count, phenoloxidase activity, respiratory burst, and phagocytic activity to V. parahaemolyticus after 24, 72 and 120 h. The phenoloxidase activity and phagocytic activity decreased significantly, whereas respiratory burst increased significantly in abalone transferred to 32 degrees C. It is concluded that transfer of abalone from 28 degrees C to 32 degrees C reduced their innate immunity and resistance against V. parahaemolyticus infection.     

The immune response of Taiwan abalone Haliotis diversicolor supertexta and its susceptibility to Vibrio parahaemolyticus at different salinity levels

Addition of NaCl at 2.5% to 3.5% to tryptic soy broth (TSB) significantly increased the growth of Vibrio parahaemolyticus. Taiwan abalone Haliotis diversicolor supertexta held in 30 per thousand seawater were injected with V. parahaemolyticus grown in TSB containing NaCl at 0.5, 1.5, 2.5, 3.5 and 4.5% at a dose of 1.6 x 10(5)colony-forming units (cfu) abalone(-1). After 48 h, the cumulative mortality was significantly higher for the abalone challenged with V. parahaemolyticus grown in 2.5% than those grown in 0.5 and 1.5% NaCl. In other experiments, abalones held in 30 per thousand seawater were injected with TSB-grown V. parahaemolyticus (1.6 x 10(5)cfu abalone(-1)), and then transferred to 20, 25, 30 and 35 per thousand seawater. All abalones held in 20 per thousand were killed in 48 h. The mortality of V. parahaemolyticus-injected abalone held in 30 per thousand was significantly lower over 24-120 h. Abalone held in 30 per thousand seawater and then transferred to 20, 25, 30 and 35 per thousand were examined for THC (total haemocyte count), phenoloxidase activity, respiratory burst, phagocytic activity and clearance efficiency of V. parahemolyticus after 24 and 72 h. The THC increased directly related with salinity levels. Phenoloxidase activity, respiratory burst, phagocytic activity and clearance efficiency of V. parahaemolyticus decreased significantly for the abalone in 20, 25 and 35 per thousand. It is concluded that the abalone transferred from 30 per thousand to 20, 25 and 35 per thousand had reduced immune ability and decreased resistance against V. parahaemolyticus infection.     

Morphologic, cytometric and functional characterisation of abalone (Haliotis tuberculata) haemocytes

This work presents the first detailed microscopic and functional analysis of the haemocytes of an abalone; the European Haliotis tuberculata. It is shown that in contrast to the situation in bivalves, only very few basophilic "granulocytes" could be found and exclusively with a histological stain. Neither flow cytometry, phase contrast observation nor transmission electron microscopy were able to detect any granular cells. The large majority of cells was constituted of "hyalinocytes", which could be sorted by flow cytometry, for the first time, into small (blast-like) and large cells. This permits a detailed analysis of haemocytes and especially of the lowly represented blast-like cells. The differences in haemolymph cell composition between bivalves and gastropods is reviewed in depth and discussed in view of the new data we present. Most of the abalone haemocytes analysed harbour many vacuoles, large glycogen deposits, lipid inclusions and acidic compartments. However, although the number of these "inclusions" was rather variable in between individual hyalinocytes, these experiments did not allow to discern subpopulations using these criteria, and the population appears more as a "differentiation continuum". Haemocytes adhere very rapidly and are immunologically active as they quickly phagocytose latex beads and zymozan particles. This study is the first step towards understanding the H. tuberculata immune system by adapting new tools to gastropods and in providing a first detailed morpho-functional study of their haemocytes.     

Expression of biomineralisation genes in tissues and cultured cells of the abalone Haliotis tuberculata

Mollusc shell biomineralisation involves a variety of organic macromolecules (matrix proteins and enzymes) that control calcium carbonate (CaCO₃) deposition, growth of crystals, the selection of polymorph, and the microstructure of the shell. Since the mantle and the hemocytes play an important role in the control of shell formation, primary cell cultures have been developed to study the expression of three biomineralisation genes recently identified in the abalone Haliotis tuberculata: a matrix protein, Lustrin A, and two carbonic anhydrase enzymes. Mantle cells and hemocytes were successfully maintained in primary cultures and were evaluated for their viability and proliferation over time using a semi-automated assay (XTT). PCR and densitometric analysis were used to semi-quantify the gene expression and compare the level of expression in native tissues and cultured cells. The results demonstrated that the three genes of interest were being expressed in abalone tissues, with expression highest in the mantle and much lower in the hemocytes and the gills. Biomineralisation genes were also expressed significantly in mantle cells, confirming that primary cultures of target tissues are suitable models for in vitro investigation of matrix protein secretion.     

Influence of temperature and spawning effort on Haliotis tuberculata mortalities caused by Vibrio harveyi: an example of emerging vibriosis linked to global warming

Since 1998, Haliotis tuberculata mass mortalities have been occurring regularly in wild abalone populations in France during their reproductive period and in conjunction with seawater summer temperature maxima and Vibrio harveyi presence. To confirm the importance of bacterial exposure, temperature and reproductive status on abalone susceptibility, experimental infections via bath exposure were performed using abalone ranging from immature to reproductively mature. Ripe abalone were more susceptible to the bacterium than immature specimens ( P <0.001), and a difference of only 1 °C in temperature had a highly significant impact on the mortalities ( P <0.001). The natural mortalities that were surveyed during summer 2007 confirmed that recent epidemic losses of European abalone appeared in conjunction with host reproductive stress, elevated temperatures and presence of the pathogen V. harveyi . In view of the elevation of the mean summer temperatures observed in Brittany and Normandy over the last 25 years, this temperature-dependent vibriosis represents a new case of emerging disease associated with global warming.     

Pathogenic Vibrio harveyi,in contrast to non‐pathogenic strains,intervenes with the p38 MAPK pathway to avoid an abalone haemocyte immune response

Vibrio harveyi is a marine bacterial pathogen responsible for episodic abalone epidemics associated with massive mortalities in France, Japan, and Australia. The aim of this study was the understanding of a possible role of the p38 MAPK in abalone haemocyte responses towards this bacterium. First, the pathogenicity of different V. harveyi strains was compared in both immersion and injection trials, and clear differences were detected. The three strains, ORM4, 04/092, and 05/053, all isolated from moribund abalone, induced up to 80% mortalities in immersion or injection challenges (LD₅₀ (ORM4) = 2.5 × 10² CFU animal^?1). The two strains, LMG 4044T and LMG 7890 were non‐pathogenic towards abalone in immersion trials, and needed very high numbers for killing by intramuscular injections (LD₅₀ = 8.9 × 10⁴ and 1.6 × 10⁵ CFU animal^?1, respectively). To start unraveling the mechanism explaining these differences, the p38‐MAPK, a keyplayer in antimicrobial immune response, was studied. The non‐pathogenic strain, LMG 7890 can be eliminated by abalone haemocytes and induces haemocyte phagocytosis and high ROS production. With different concentrations of a p38‐specific inhibitor, SB203580, p38 implication was shown. This inhibitor reduced phagocytosis and ROS induction leading to LMG 7890 proliferation. In the case of the pathogenic ORM4 which can not be eliminated by abalone haemocytes, no phagocytosis and ROS production was induced, and a retarded p38 activation was observed. Taken together, our results suggest that p38 MAPK modulation may be one of the ways of virulent V. harveyi to attack its host and escape abalone immune response. J. Cell. Biochem. 106: 152–160, 2009. © 2008 Wiley‐Liss, Inc.     

Influence of elevated temperatures on the immune response of abalone, Haliotis rubra

Elevated water temperature can act as a stressor impacting the immune responses of molluscs, potentially increasing their susceptibility to microbial infections. Abalone are commercially important marine molluscs that have recently experienced disease outbreaks caused by a herpesvirus and Vibrio bacteria. Sampling of wild-caught Haliotis rubra showed a significant correlation between water temperature and both antiviral and antibacterial activity, with higher activity in summer than in winter months. However, antibacterial activity was compromised in favour of antiviral activity as the water temperatures peaked in summer. A controlled laboratory experiment was then used to investigate several immune responses of H. rubra, including total haemocyte count (THC), stimulated superoxide anion production (SO), antiviral activity against a model herpesvirus, herpes simplex virus type 1 and antibacterial activity against a representative pathogenic bacterium, Vibrio anguillarum, over one week after raising water temperature from 18 to 21 or 24 °C. THC and SO increased at day 1 and then dropped back to control levels by days 3 and 7. By comparison, the humoural immune parameters showed a delayed response with antibacterial and antiviral activity significantly increasing on days 3 and 7, respectively. Consistent with the field study, antibacterial activity became significantly depressed after prolonged exposure to elevated temperatures. A principal components analysis on the combined immune parameters showed a negative correlation between antiviral and antibacterial activity. SO was positively correlated to THC and neither of these cellular parameters were correlated to the humoural antimicrobial activity. Overall, this study indicates that abalone may have more resilience to viruses than bacterial pathogens under conditions of elevated temperature, such as those predicted under future climate change scenarios.     

Virulence of Vibrio parahaemolyticus isolated from cultured small abalone, Haliotis diversicolor supertexta, with withering syndrome

Outbreaks of mass mortality among cultured small abalone Haliotis diversicolor supertexta with withering syndrome occurred in May and September 1998 in Kao-Hsiung, Taiwan. Bacterial strains CH-1 and B4 were isolated from the haemolymph of the moribund small abalone using tryptic soy agar supplemented with 3% NaCl and/or thiosulphate citrate bile salt sucrose agar. These two strains were characterized and identified as Vibrio parahaemolyticus on the basis of various biochemical tests. The B4 strain and its extracellular products were virulent to small abalone with LD(50) values of 1.6 x 10(5) colony-forming units and 7.58 microg protein g-1 body weight, respectively.     

Adaptation and increased susceptibility to infection associated with constitutive expression of misfolded SP-C

Mutations in the gene encoding SP-C (surfactant protein C; SFTPC) have been linked to interstitial lung disease (ILD) in children and adults. Expression of the index mutation, SP-C(Deltaexon4), in transiently transfected cells and type II cells of transgenic mice resulted in misfolding of the proprotein, activation of endoplasmic reticulum (ER) stress pathways, and cytotoxicity. In this study, we show that stably transfected cells adapted to chronic ER stress imposed by the constitutive expression of SP-C(Deltaexon4) via an NF-kappaB-dependent pathway. However, the infection of cells expressing SP-C(Deltaexon4) with respiratory syncytial virus resulted in significantly enhanced cytotoxicity associated with accumulation of the mutant proprotein, pronounced activation of the unfolded protein response, and cell death. Adaptation to chronic ER stress imposed by misfolded SP-C was associated with increased susceptibility to viral-induced cell death. The wide variability in the age of onset of ILD in patients with SFTPC mutations may be related to environmental insults that ultimately overwhelm the homeostatic cytoprotective response.     

Changes to the phenotypic profile of Vibrio harveyi when infected with the Vibrio harveyi myovirus-like (VHML) bacteriophage

AIMS: To determine if infection of Vibrio harveyi with the V. harveyi myovirus-like (VHML) bacteriophage causes a change to the phenotypic profile of this species. METHODS AND RESULTS: Using 46 biochemical and metabolic tests, phenotypic profiles for noninfected V. harveyi and VHML infected V. harveyi were developed. Comparison of the infected and bacteriophage-infected strains of V. harveyi 645, 20 and 45 were found to have different test results for d-gluconate utilization, gamma-glutamyl transpeptidase and sulfatase activity, respectively. Using probabilistic identification, VHML infected and noninfected strains were identified as V. harveyi and had similar Willcox probability scores though the modal likelihood scores were reduced for VHML infected strains. One VHML infected strain, 642b, was misidentified as V. campbellii by phenotyping but not by PCR. It would appear that the phenotype of V. harveyi strains infected with VHML, are sufficiently altered that they occur at the margins of the known range of strain variation for V. harveyi. CONCLUSION: Infection of V. harveyi with VHML causes the phenotypic profile of the bacterium to change. This change reduces the modal likelihood score resulting in a poorer level of assurance for an identification of V. harveyi, especially in the natural host, strain 642. The bacteriophage VHML integrates into different sites in different strains of V. harveyi. SIGNIFICANCE AND IMPACT OF THE STUDY: The identification of V. harveyi as the causative agent of mortality in aquatic organisms is predominantly achieved through phenotyping. Since bacteriophages alter virulence in V. harveyi, understanding the effect they have on phenotype is important.     

Effects of dietary pyridoxine on immune responses in abalone, Haliotis discus hannai Ino

A feeding experiment was conducted to investigate the effects of dietary pyridoxine (PN) on the immune responses of abalone, Haliotis discus hannai Ino. Purified diets supplemented with 0, 40, 800 mg PN kg(-1) or 80 mg kg(-1) of 4-deoxypyridoxine (PN antagonist) were fed to adult abalone (initial weight 45.77 +/- 0.25 g; initial shell length 68.02 +/- 0.78 mm) for 90 days. The air-dried brown kelp, Laminaria japonica, was used as a control diet. Each diet was fed to three replicate groups of abalone in a recirculation system using a completely randomised design. The results showed that weight gain ratio (WGR) of the abalone generally increased with the level of dietary PN supplementation though no significant differences were found among the treatments (P > 0.05). Phagocytic and phenoloxidase activities were significantly higher in abalone fed diets supplemented with 800 mg PN kg(-1) than those fed the PN-free diet or the one with 4-deoxypyridoxine (P < 0.05). Agglutination titre and respiratory burst activity were significantly higher in abalone fed diets supplemented with 40 mg PN kg(-1) than those fed the PN-free diet or the one with 4-deoxypyridoxine (P < 0.05). There were no significant differences in immunological characteristics between the abalone fed the diet containing 40 mg PN kg(-1) and those fed the diet containing 800 mg PN kg(-1) (P > 0.05). L. japonica resulted in significantly lower agglutination titre, respiratory burst and phagocytic activities than the artificial diets supplemented with 40 or 800 mg PN kg(-1) (P < 0.05). Total haemocyte count (THC), serum protein concentration, and the activities of lysozyme and acid phosphatase were not significantly affected by the dietary treatments (P > 0.05). These results demonstrate that dietary deficiency of pyridoxine suppresses the immune functions in H. discus hannai, and further investigations are needed to optimise the dietary level of this vitamin for maintaining the best immune responses in abalone.     

Ultrastructural developmental cycle of Haplosporidium montforti (phylum Haplosporidia) in its farmed abalone host, Haliotis tuberculata (Gastropoda)

The sequential developmental cycle of Haplosporidium montforti, a recently described species from farmed abalone Haliotis tuberculata (Gastropoda), was studied. Ornamented and operculated mature spores were electron dense. The nucleus of the uninucleated free cell divided successively, giving rise to multinucleate plasmodia, containing up to 100-120 nuclei. Later, the plasmodia developed into sporonts inside sporocysts with irregular contours. Each of their nuclei gave rise to uninucleate sporoblasts. At the next phase of development, a very irregular membranous group of cisternae began to differentiate in the cytoplasm of each sporoblast, surrounding each nucleus and the adjacent cytoplasm. Each sporoblast differentiated into a spore. This process was characterized by the appearance of dense blisters of amorphous material at the periphery that gradually formed the prespore wall and pre-operculum. Simultaneously, in the endosporoplasm, the spherulosome and several haplosporosomes were formed. During the final phase of the maturation process, the spores became gradually denser, and the endosporoplasmic structures were barely visible.