Mauro Meloni

Molecular Characterization of Anisakis Larvae from Fish Caught Off Sardinia

abstract:  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.

A sensitive FRET probe assay for the selective detection of Mycobacterium marinum in fish

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.

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 102 env fragments/PCR) with a linear dynamic range of quantitation from 103 to 107 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.

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.