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Antimicrobial polypeptides (AMPPs) are humoral components of the vertebrates and invertebrates innate immune system. Their potent broad spectrum antimicrobial activities have drawn the attention of the scientific community to their potential use not only as an alternative to antibiotics but also as functional targets for immunostimulants in order to enhance the host immunity. Fish synthesize a great number of these peptides but in European sea bass, an important fish species in the Mediterranean aquaculture, only a few AMPPs have been studied and these surveys have highlighted their functional role as predictive markers of stressful conditions. Many aspects concerning AMPP mode of action in the host during bacterial infections are still unknown. In this work a 72 h time course experiment, performed on juvenile sea bass intraperitoneally (i.p.) injected with a sub-lethal dose of Vibrio anguillarum, was aimed to investigate the mRNA expression of four specific AMPP genes and interleukin-1β (IL-1β) in skin, gills, spleen, and head kidney. AMPP genes were: dicentracin (DIC), histone-like protein 1 (HLP-1), histone-like protein 2 (HLP-2) and hemoglobin-like protein (Hb-LP). The delta-delta C(T) method in real-time RT-PCR allowed to gain more knowledge about temporal dynamics, preferential sites of expression as well as immunological and physiological role of these molecular markers. DIC was significantly up-regulated mainly in head kidney at 1.5-3 h post-infection (p.i.). HLP-1 showed an extended-time overexpression in gills and a significant up-regulation in spleen. HLP-2 was interestingly overexpressed in gills at 24 h p.i., while Hb-LP showed a significant up-regulation in skin for all the 72 h trial as well as lower but always significant values either in gills or in spleen. Different was the response of IL-1β that showed a dramatic up-regulation in spleen and head kidney at 8 h p.i. whilst in gills it displayed a severe inhibition. During this survey the i.p. stimulus surely conditioned the AMPP expression in skin and gills, especially as regards the DIC that as piscidin-related gene has an important defensive role in the mucosal tissues. However, two unconventional AMPP genes such as HLP-2 and Hb-LP, strictly related to the physiological mechanisms of fish, were less affected in terms of expression by the route of infection, being more evident in peripheral loci. These findings might suggest them as potential markers to be analyzed within plans of health survey in fish farms.

Anisakis spp. larvae are parasitic, and potentially zoonotic, nematodes transmitted by marine fish and cephalopods, which are the main intermediate hosts of the third larval stage. The accidental consumption of infected raw or poorly cooked fish may cause gastroenteric diseases and allergies in humans. The aim of the present study was to use polymerase chain reactionrestriction fragment length polymorphism (PCR-RFLP) to define the occurrence, species variability, and host preferences of Anisakis spp. larvae in fish caught off the coast of Sardinia. Necropsy was used on 285 samples; 552 Anisakis spp. L3 larvae were isolated from 87 fish that tested positive for this nematode. Anisakis pegreffii was most frequently encountered (90.6%), with a primary preference for Scomber scombrus, Zeus faber, and Trachurus mediterraneus. In contrast, the prevalence of Anisakis physeteris was only 1.3%. A hybrid genotype of Anisakis simplex sensu stricto and Anisakis pegreffii was also observed, which confirms the results of previous studies carried out in the western Mediterranean. Interestingly, no Anisakis simplex s.s. larvae were recovered. These results indicate that the diversity of Anisakis species is low in Sardinia waters, probably because of its geographic position. © 2011 American Society of Parasitologists.

Mycobacterium marinum is the causative agent of mycobacteriosis in wild and cultured fish and of atypical infection in humans. For the diagnosis of M. marinum, cultural and traditional polymerase chain reaction (PCR) methods are currently used. However, these protocols, although able to discriminate within Mycobacterium spp., have proved to be time-consuming or difficult to carry out. For this reason, the aim of this study was to obtain a rapid and specific diagnostic tool to quantify fish Mycobacterium spp. or to discriminate M. marinum from other mycobacteria. A primary PCR amplification with SYBR Green had a detection limit (dl) of 10(2)Mycobacterium DNA copies with a log-linear quantification range up to 10(4) (R(2) = 0.99). The second PCR using FRET probes, flanking a region containing species specific nucleotide variations, was designed and validated with synthetic erp gene fragments corresponding to different mycobacterial species, different whole mycobacteria suspensions, experimentally infected fish tissues, tissues from experimentally infected fish, and samples of cultured fish. The results show that the FRET probes demonstrate a high specificity as the melting curve analysis allowed efficient discrimination of M. marinum from Mycobacterium chelonae, Mycobacterium fortuitum, Mycobacterium pseudoshottsii, Mycobacterium shottsii and Mycobacterium ulcerans. The kidney is the organ with the strongest detection signal and using fish tissues the method has a mean sensitivity of 50 DNA copies/PCR.

Bluetongue virus (BTV) is the causative agent of Bluetongue (BT) disease in ruminant livestock and occurs almost worldwide between latitudes 35 degrees S and 50 degrees N; 24 serotypes of BTV are known of which 8 circulate periodically within parts of the Mediterranean Region. A fast (about 3.5 h) and versatile diagnostic procedure able to detect and quantify BTV-RNA, has been developed using a Molecular Beacon (MB) fluorescent probe; PCR primers were designed to target 91 bp within the NS3 conserved region of the viral RNA segment 10 (S10) and bracketed the MB fluorescence probe hybridisation site. The MB fluorescent probe was used to develop two Bluetongue serogroup-specific assays: a quantitative real time reverse transcriptase polymerase chain reaction (RT-PCR) and a traditional RT-PCR. These were tested using BTV-RNAs extracted from the blood and organs of BT-affected animals, and from virus isolate suspensions. The samples included ten serotypes (BTV-1-BTV-9 and BTV-16); of these, BTV serotypes -1, -2, -4, -9 and -16 have since 1998 been involved in the extensive outbreaks of BT across the Mediterranean Region. To evaluate the specificity and sensitivity of the MB probe, all positive samples (and negative controls) were tested using the developed quantitative real time RT-PCR and traditional RT-PCR assays. The former test had a detection limit of 10(3) cDNA molecules per reaction with a log-linear quantification range of up to 10(11) (R2 = 0.98), while the latter test was able to detect 500 cDNA-BTV molecules/PCR. The results show that the MB fluorescent probe is both rapid and versatile for the laboratory diagnosis of Bluetongue and for quantifying levels of viraemia in BTV-affected animals. An "in silico" comparison of the primers and MB fluorescent probe used in this study showed that it is possible to detect all 24 serotypes of BTV.