Pietro Giorgio Tiscar

Piezoelectric Sensors Based on Biomimetic Peptides for the Detection of Heat Shock Proteins (HSPs) in Mussels

Abstract We describe a set of label‐free piezoelectric biosensors for fast, qualitative, and direct detection of HSP 70 in crude extract of the mussel mantle. A monoclonal anti‐HSP 70 antibody and three heptapeptides chosen using a computational/combinatorial approach were immobilized covalently to gold particles on quartz surfaces using a self‐assembled monolayer (SAM). A clear frequency shift in the presence of standard solutions of HSP 70 from bovine brain was obtained. The difference in signal intensity among the biomimetic sensors (the peptide sensors) was nicely correlated with the computationally predicted binding scores. Moreover, the peptides exhibited a signal comparable to the monoclonal antibody based immunosensor. Regeneration of the affinity sensors was also investigated obtaining a significant loss of signal only after 10 regeneration cycles with repeatability in the 20%–28% range and reproducibility in the 25%–35% range. Cross‐reactivity of the HSP sensors was tested using bovine serum albumin (BSA) and rabbit IgG. All the affinity sensors exhibited low nonspecific binding, compared with a blank sensor and a negative control sensor. The sensitivity pattern with the real sample (mussel mantle) reflected the one obtained from the standard solution of HSPs 70, demonstrating that all the sensors are able to detect the target analyte in a complex matrix as in a crude extract.

Innovative vaccination protocol against vibriosis in Dicentrarchus labrax (L.) juveniles: Improvement of immune parameters and protection to challenge

The effect of vaccination on immune parameters of European sea bass, Dicentrarchus labrax, is not fully established, as well as surveyed throughout rearing till the commercial size. Furthermore, available information on the possible role of booster treatments is scarce. Sea bass juveniles were vaccinated against Listonella anguillarum using a commercial bivalent formulation administered by immersion (priming: 95 dph; booster: 165 dph) or by immersion (priming: 95 dph; booster: 165 dph) and subsequent i.p. injection (booster: 233 dph). Serum specific IgM and numbers of IgM(+) cells in head kidney and spleen evidenced B-cell responses mainly after the immersion booster, accompanied by increased TcR-β transcripts and leucocyte respiratory burst. Immune enhancement was confirmed by the protection towards i.p. challenges with a virulent strain. RPS accounted for >70% in fish immersion-boosted and near 100% in fish further boosted i.p. Differently from usual farm practices, this innovative vaccination protocol proved to be highly effective. Booster treatments are therefore strongly recommended.

Effects of high temperature and exposure to air on mussel (Mytilus galloprovincialis, Lmk 1819) hemocyte phagocytosis: modulation of spreading and oxidative response.

Hemocytes are a critical component of the mussel defense system and the present study aims at investigating their spreading and oxidative properties during phagocytosis under in vivo experimental stress conditions. The spreading ability was measured by an automated cell analyzer on the basis of the circularity, a parameter corresponding to the hemocyte roundness. The oxidative activity was investigated by micromethod assay, measuring the respiratory burst as expression of the fluorescence generated by the oxidation of specific probe. Following the application of high temperature and exposure to air, there was evidence of negative modulation of spreading and oxidative response, as revealed by a cell roundness increase and fluorescence generation decrease. Therefore, the fall of respiratory burst appeared as matched with the inhibition of hemocyte morphological activation, suggesting a potential depression of the phagocytosis process and confirming the application of the circularity parameter as potential stress marker, both in experimental and field studies.

Age related properties of the Adriatic clam Chamelea gallina (L. 1758) hemocytes

The clam Chamelea gallina (L 1758) represents an important shellfish resource along Mediterranean coasts and its progressive depletion has been ascribed both to the overexploitation of stocks and to environmental or anthropic stressors. In this context, the investigation on immune parameters could represent a valid approach to measure the clam homeostasis condition and its possible influence on population dynamics. On this basis, the innate immune system, mainly represented by hemocyte phagocytosis, was investigated in organisms of different size. The results indicated a better phagocytic response in larger clams, strictly related to a greater concentration of granulocytes. A such variation in hemolymph composition appeared not dependent on environmental or endogenous factors, but rather on clam aging.

Defensive response of European sea bass (Dicentrarchus labrax) against Listonella anguillarum or Photobacterium damselae subsp. piscicida experimental infection.

Sea bass were experimentally infected with Listonella anguillarum or Photobacterium damselae subsp. piscicida (Phdp). At 24 and 72h post-infection, the expression analysis of immune-relevant genes (IL-1β, IL-6, IL-8, Hepcidin), the transcriptional level and detection of HSP70, and the quantification of serum iron were investigated in association with the histological analysis and the bacterial recognition in tissues by immunohistochemistry. At 15 days post-infection, the specific antibody response was detected in surviving fish, as well as the transcriptional levels of TcR and BcR sequences. Both experimental infections were characterized by a similar acute response, whereas different histological and immunohistochemistry evidences were observed. In particular, the early reaction appeared suitable for the clearance of L. anguillarum, thus limiting the histological lesions, the bacterial dissemination and the further development of acquired immunity in surviving fish. On the contrary, the innate response appeared not enough to resolve the Phdp infection, which was characterized by tissue damage, bacterial widespread and substantial detection of specific humoral immunity in surviving fish, also associated to lymphocytes clonal expansion. Besides the opportunistic conditions involved in fish vibriosis and pasteurellosis, the comparison between these experimental infection models seems to suggest that the rate of development of the acquired immunity is strictly linked to the activation of the host innate response combined to the degree of bacterial virulence.

Variability of the hemocyte parameters of cultivated mussel Mytilus galloprovincialis (Lmk 1819) in Sabaudia (Latina, Italy) coastal lagoon.

The Sabaudias lake consists of a protected coastal lagoon, located in the central Italy, historically characterized by recurrent mortality events of marine fauna during warmer months. A field study was monthly conducted on mussels Mytilus galloprovincialis cultivated inside the lagoon, measuring hemocyte parameters as total circulating count (THC), viability (HV), spreading and oxidative response to in vitro phagocytosis stimulation. A depression of the immune response was observed during the spring season, as indicated by higher values of hemocyte circularity and lower luminescence levels related to respiratory burst, also associated to modulation of THC and HV. The water temperature and the oxygen concentration appeared as the major environmental factors having influence on the phagocytosis activity. Therefore, the hemocyte variations have been intended as early danger signal to evaluate the immunodepression induced by the environmental stressors which could reveal in advance the development of critical situations for mussel survival.

Microbial Diseases of Bivalve Mollusks: Infections, Immunology and Antimicrobial Defense

A variety of bivalve mollusks (phylum Mollusca, class Bivalvia) constitute a prominent commodity in fisheries and aquacultures, but are also crucial in order to preserve our ecosystem’s complexity and function. Bivalve mollusks, such as clams, mussels, oysters and scallops, are relevant bred species, and their global farming maintains a high incremental annual growth rate, representing a considerable proportion of the overall fishery activities. Bivalve mollusks are filter feeders; therefore by filtering a great quantity of water, they may bioaccumulate in their tissues a high number of microorganisms that can be considered infectious for humans and higher vertebrates. Moreover, since some pathogens are also able to infect bivalve mollusks, they are a threat for the entire mollusk farming industry. In consideration of the leading role in aquaculture and the growing financial importance of bivalve farming, much interest has been recently devoted to investigate the pathogenesis of infectious diseases of these mollusks in order to be prepared for public health emergencies and to avoid dreadful income losses. Several bacterial and viral pathogens will be described herein. Despite the minor complexity of the organization of the immune system of bivalves, compared to mammalian immune systems, a precise description of the different mechanisms that induce its activation and functioning is still missing. In the present review, a substantial consideration will be devoted in outlining the immune responses of bivalves and their repertoire of immune cells. Finally, we will focus on the description of antimicrobial peptides that have been identified and characterized in bivalve mollusks. Their structural and antimicrobial features are also of great interest for the biotechnology sector as antimicrobial templates to combat the increasing antibiotic-resistance of different pathogenic bacteria that plague the human population all over the world.

Genetic diversity of Arcobacter isolated from bivalves of Adriatic and their interactions with Mytilus galloprovincialis hemocytes

The human food‐borne pathogens Arcobacter butzleri and A. cryaerophilus have been frequently isolated from the intestinal tracts and fecal samples of different farm animals and, after excretion, these microorganisms can contaminate the environment, including the aquatic one. In this regard, A. butzleri and A. cryaerophilus have been detected in seawater and bivalves of coastal areas which are affected by fecal contamination. The capability of bivalve hemocytes to interact with bacteria has been proposed as the main factor inversely conditioning their persistence in the bivalve. In this study, 12 strains of Arcobacter spp. were isolated between January and May 2013 from bivalves of Central Adriatic Sea of Italy in order to examine their genetic diversity as well as in vitro interactions with bivalve components of the immune response, such as hemocytes. Of these, seven isolates were A. butzleri and five A. cryaerophilus, and were genetically different. All strains showed ability to induce spreading and respiratory burst of Mytilus galloprovincialis hemocytes. Overall, our data demonstrate the high genetic diversity of these microorganisms circulating in the marine study area. Moreover, the Arcobacter–bivalve interaction suggests that they do not have a potential to persist in the tissues of M. galloprovincialis.

Polymorphonuclear cells and reactive oxygen species in contagious bovine pleuropneumonia: New insight from in vitro investigations

Reactive oxygen species (ROS) are suggested to play a role in the pathogenesis of contagious bovine pleuropneumonia, a severe respiratory disorder caused by Mycoplasma mycoides subsp. mycoides (Mmm). The present study investigated the generation of ROS by different strains of Mmm, as well as their effect on the oxidative response of bovine neutrophils. The production of ROS was indirectly measured using a luminol-based chemiluminescence assay. Our results confirm that Mmm can produce ROS via the metabolism of glycerol, significant differences existing between African and European strains. Mmm was capable of adhering to the external surface of neutrophils. Interestingly, Mmm enhanced the respiratory burst of bovine neutrophils. This activity was particularly pronounced with the African field strain and in presence of glycerol. Taken together, our data argue in favour of a major role for neutrophils as the main source of ROS in contagious bovine pleuropneumonia.