Recovery of Staphylococcus aureus in Gray Mugil cephalus Roe (Bottarga): Investigation by an Integrated Cultural/Molecular Approach.

In the Mediterranean area, salted and dried roe from the gray Mugil cephalus "bottarga" represent a speciality food with great commercial value. Bottarga is currently produced by a traditional handmade process and, the risk of human bacterial contamination during its manufacturing is still unknown; in this perspective the foodborne pathogen Staphylococcus aureus could potentially contaminate this product due to poor sanitation or bad handling during processing. The aim of this work is: to evaluate the contamination level of foodborne pathogens at different product manufacturing stages and, in addition, to describe a fast and realizable method for the rapid detection of S. aureus in bottarga samples in the field. A cultural procedure was initially used to investigate the occurrence of S. aureus and the other main foodborne pathogens in bottarga samples at the different manufacturing stages (from roe to final product). In addition, a molecular approach was used to rapidly determine the presence of total bacteria, S. aureus, and its potential toxigenicity. Of the 194 specimens analyzed, we identified: Clostridium perfringens, Enterococcus spp. and Enterobacteriaceae. However, some samples resulted as being contaminated with S. aureus (4% in roe and 8.7% in the final product). During the bottarga manufacturing process, we observed an increase in pathogen levels (from 10(2) to 10(5) CFU/g) in contaminated samples, and entA and entB genotypes were identified. Reconstruction experiments suggest that the fresh roe and the bottarga (not completely dried) could represent a risk for the contamination and growth of pathogen bacteria.

Preliminary study on expression of antimicrobial peptides in European sea bass (Dicentrarchus labrax) following in vivo infection with Vibrio anguillarum. A time course experiment.

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-1ß (IL-1ß) 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-1ß 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.

Presence of Contracaecum spp. in teleosts cultured and fished in Sardinia.

This study reports the results of the finding of Contracaecum spp. during a survey on endoparasites isolated from cultured and wild fish and also from some cephalopods caught in Sardinian waters. Contracaecum spp. is a nematode belonging to the Anisakidae, and is reported to cause zoonosis in humans. Nematodes were detected after visual inspection and enzymatic digestion and then identified by morphologic observation, which was confirmed by PCR. The results show that Contracaecum spp. were found in both fish caught from sea or lagoon, and in both cultured and wild fish: 33 of the parasitized samples were wild fish (24 caught in the sea and 9 in lagoons) and 11 were cultured ones. The prevalence of Contracaecum spp. was higher in Diplodus spp. (16.0%), Sparus aurata (15.8%) and Mullus spp. (14.6%). Larvae were also found by enzymatic digestion at muscular level in 5 species, with the highest prevalence in S. aurata (10.5%). The results of this study indicate that Contracaecum spp. was present in cultured fish such as S. aurata, Diplodus spp. and Dicentrarchus labrax. All cultured fish with parasites were collected from land-based semi-intensive tanks whose water came from an adjacent lagoon. Finally, the evidence that this parasite is found in both cultured and wild fish leads us to re-consider the zoonotic potential of Contracaecum spp., in particular when one bears in mind its dimensions at the L3 stage, when it is barely visible to the human eye.

Development and Field Testing of a Real-Time PCR Assay for Caprine Arthritis-Encephalitis-Virus (CAEV).

Caprine arthritis/encephalitis (CAE) of goats and occasionally sheep are persistent virus infections caused by a lentivirus (CAEV). This viral infection results in arthritis in adult animals and encephalitis in kids. Prognosis for the encephalitic form is normally poor, with substantial economic loss for the farm. In this context an early/fast laboratory diagnosis for CAEV infection could be useful for effective prophylactic action. In this work we performed a quantitative real time PCR designed on the CAEV env gene to detect/quantify in goat/sheep samples, viral RNA or proviral DNA forms of CAEV. This procedure was validated in 15 sheep, experimentally infected with CAEV or with a highly correlated lentivirus (visna maedi, MVV); in addition, a total of 37 clinical goat specimens recruited in CAEV positive herds were analyzed and compared using serological analysis (Elisa and AGID). All samples infected with MVV resulted negative. In sheep experimentally infected with CAEV, proviral DNA was detectable 15 days post infection, whereas the serological methods revealed an indicative positivity after 40-60 days.This method showed a sensitivity of 10(2) env fragments/PCR) with a linear dynamic range of quantitation from 10(3) to 10(7)env fragments/PCR; the R2 correlation coefficient was 0.98. All subjects with a clinical diagnosis for Caprine Arthritis-Encephalitis (CAE) resulted CAEV DNA positive.

Prevalence of Anisakis spp. and Hysterothylacium spp. larvae in teleosts and cephalopods sampled from waters off Sardinia.

A study was carried out on the presence of Anisakis and Hysterothylacium larvae in fish and cephalopods caught in Sardinian waters. A total of 369 specimens of 24 different species of teleosts and 5 species of cephalopods were collected from different fishing areas of Sardinia. Larvae were detected and isolated by both visual inspection and enzymatic digestion. These methods allowed Anisakis type I and type II third-stage larvae and Hysterothylacium third- and fourth-stage larvae to be detected. The prevalence, mean intensity, and mean abundance were calculated. The results obtained showed the highest prevalence of Anisakidae in Zeus faber (100%) and of Anisakis in Micromesistius poutassou (87.5%). The highest prevalence of Anisakis type I larvae was in M. poutassou (81.2%), and that of Anisakis type II larvae was in Todarodes sagittatus (20%). The highest values for prevalence, mean intensity, and mean abundance for Hysterothylacium were found in Z. faber. These prevalences and the mean intensity and abundance were higher than those reported by different authors in other Mediterranean areas. This may be because the enzymatic digestive method used in this research resulted in higher recovery levels. The data suggest that Sardinia may be a high-risk area for zoonotic diseases and that measures such as information campaigns, aimed at both sanitary service personnel and consumers, should be employed to limit the spread of such zoonosis.

Molecular characterization of Anisakis larvae from fish caught off Sardinia.

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 reaction-restriction 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.

Influence of Moraxella sp. colonization on the kidney proteome of farmed gilthead sea breams (Sparus aurata, L.).

BACKGROUND: Currently, presence of Moraxella sp. in internal organs of fish is not considered detrimental for fish farming. However, bacterial colonization of internal organs can affect fish wellness and decrease growth rate, stress resistance, and immune response. Recently, there have been reports by farmers concerning slow growth, poor feed conversion, and low average weight increase of fish farmed in offshore floating sea cages, often associated with internal organ colonization by Moraxella sp. Therefore, presence of these opportunistic bacteria deserves further investigations for elucidating incidence and impact on fish metabolism. RESULTS: A total of 960 gilthead sea breams (Sparus aurata, L.), collected along 17 months from four offshore sea cage plants and two natural lagoons in Sardinia, were subjected to routine microbiological examination of internal organs throughout the production cycle. Thirteen subjects (1.35%) were found positive for Moraxella sp. in the kidney (7), brain (3), eye (1), spleen (1), and perivisceral fat (1). In order to investigate the influence of Moraxella sp. colonization, positive and negative kidney samples were subjected to a differential proteomics study by means of 2-D PAGE and mass spectrometry. Interestingly, Moraxella sp. infected kidneys displayed a concerted upregulation of several mitochondrial enzymes compared to negative tissues, reinforcing previous observations following lipopolysaccharide (LPS) challenge in fish. CONCLUSIONS: Presence of Moraxella sp. in farmed sea bream kidney is able to induce proteome alterations similar to those described following LPS challenge in other fish species. This study revealed that Moraxella sp. might be causing metabolic alterations in fish, and provided indications on proteins that could be investigated as markers of infection by Gram-negative bacteria within farming plants.

Rapid detection and quantitation of Bluetongue virus (BTV) using a Molecular Beacon fluorescent probe assay.

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.

Control of the expression and compartmentalization of (sigma)G activity during sporulation of Bacillus subtilis by regulators of (sigma)F and (sigma)E.

During formation of spores by Bacillus subtilis the RNA polymerase factor sigma(G) ordinarily becomes active during spore formation exclusively in the prespore upon completion of engulfment of the prespore by the mother cell. Formation and activation of sigma(G) ordinarily requires prior activity of sigma(F) in the prespore and sigma(E) in the mother cell. Here we report that in spoIIA mutants lacking both sigma(F) and the anti-sigma factor SpoIIAB and in which sigma(E) is not active, sigma(G) nevertheless becomes active. Further, its activity is largely confined to the mother cell. Thus, there is a switch in the location of sigma(G) activity from prespore to mother cell. Factors contributing to the mother cell location are inferred to be read-through of spoIIIG, the structural gene for sigma(G), from the upstream spoIIG locus and the absence of SpoIIAB, which can act in the mother cell as an anti-sigma factor to sigma(G). When the spoIIIG locus was moved away from spoIIG to the distal amyE locus, sigma(G) became active earlier in sporulation in spoIIA deletion mutants, and the sporulation septum was not formed, suggesting that premature sigma(G) activation can block septum formation. We report a previously unrecognized control in which SpoIIGA can prevent the appearance of sigma(G) activity, and pro-sigma(E) (but not sigma(E)) can counteract this effect of SpoIIGA. We find that in strains lacking sigma(F) and SpoIIAB and engineered to produce active sigma(E) in the mother cell without the need for SpoIIGA, sigma(G) also becomes active in the mother cell.