PRESENCE OF ANISAKIS PEGREFFII IN FARMED SEA BASS ( DICENTRARCHUS LABRAX L.) COMMERCIALIZED IN SOUTHERN ITALY: A FIRST REPORT

We examined 151 European sea bass ( Dicentrarchus labrax L.) samples from farms and fish markets of Sicily (Southern Italy) for Anisakidae larvae detection. All the samples were examined by visual inspection and modified chloro-peptic digestion. Two nematode larvae were found in the viscera of only one European sea bass sample from a farm located in Greece (FAO 37.3), giving a total prevalence of infestation of 0.7%. No other parasites were found after chloro-peptic digestion of the samples. The larvae were morphologically ascribed, at genus level, to morphotypes I and molecularly identified as Anisakis pegreffii . To the best if our knowledge, this is the first report on the presence of anisakid parasites in farmed European sea bass of Mediterranean Sea. Our findings suggest that the risk of exposure to Anisakidae nematodes in farmed European sea bass remains very low. However, further data on Mediterranean farms are needed to have a detailed risk analysis.

 151 farmed European sea bass samples were examined for Anisakis larvae detection  Two nematode larvae were found in the viscera of only one sample (prevalence 0.7%)  The larvae were confirmed as Anisakis pegreffii by PCR-RFLP and sequence analysis  The larvae detected belonged to a sample from farms of Greece (FAO 37.3)  This work give a first report on the presence of Anisakis in farmed European sea bass A C C E P T E D M A N U S C R I P T

INTRODUCTION
Anisakis and other nematodes belonging to the Anisakidae family are parasites with a worldwide distribution and a complex life cycle which involves several marine organisms at different trophic levels Ferrantelli et al., 2015;Mattiucci and Nascetti, 2006). Anisakids parasites are of interest for health because of their high zoonotic capability. These nematodes are known to cause clinical manifestations called Anisakiasis or Anisakidosis in humans after the consumption of raw or undercooked parasitized fish. The clinical manifestations of Anisakiasis and Anisakidosis include epigastric pain, nausea, vomiting, diarrhoea and urticaria (Mattiucci et al, 2013).
In addition, several authors confirmed the capability of certain Anisakis simplex complex proteins to induce acute allergic reactions in humans (Audicana and Kennedy, 2008). Gastroallergic reactions associated with Anisakis pegreffii infection, were reported in Italy (Mattiucci et al, 2013). In response to these findings, the European Food Safety Authority (EFSA) published a Scientific Opinion on risk assessment of parasites in fishery products (EFSA, 2010). The EFSA scientific opinion declare that the risk of infection with anisakids is negligible only for farmed Atlantic salmon reared in floating cages or onshore tanks and fed on compound feedstuffs. In this regard, most of the studies reported the absence of anisakid nematodes in farmed Atlantic and Pacific salmon (Angot and Brasseur, 1993;Lunestad, 2003). Farmed fish have not generally been regarded as infected with anisakid larvae. Apart from farmed Atlantic salmon, sufficient monitoring data are not available for any other farmed fish therefore the EFSA encourage epidemiological studies on the fish species reared in European territory. At present, few studies have focused on the prevalence of anisakid nematodes in farmed fish of Mediterranean Sea such as European Sea Bass (Dicentrarchus labrax L.) and Gilthead Sea Bream (Sparus aurata L.) (Peñalver et al., 2010). Of these, no information on infection with larval anisakids are available. However, the experimental susceptibility of Gilthead Sea Bream (Sparus aurata L.) and European bass (Dicentrarchus labrax L.) towards Anisakis has been demonstrated (Macrì et al, 2012). European Sea Bass is one of the principal fish farmed in the Mediterranean. Although seabass are farmed in seawater ponds and lagoons, the bulk of production comes from sea cage farming.
The probability of infection of the wild juvenile fish used as basis for this culture method is still unknown. In Mediterranean, the majority of the wild fish belonged to Italian and Greek coasts are infected with larvae belonging to the A. pegreffii species (Abollo et al, 2003;Chaligiannis et al, 2012;Ferrantelli et al., 2015). Given this, the present work aimed to evaluate the presence of anisakid nematodes in farmed European sea bass produced and commercialized in Southern Italy to obtain a detailed risk assessment on the presence of zoonotic anisakid larvae in farmed fish of Mediterranean.

Sampling plan and sample collection
A total of 83 European sea bass from 2 farms located in Licata (40 samples) and Pachino (43 samples The samples were stored at + 4 °C and transferred to the laboratories of the Centro di Referenza Nazionale per le Anisakiasi for the morphometric and inspection analysis.

Inspection of the samples and morphological analysis of the larvae
The fish samples were initially submitted to morphometric analysis by the calculation of maximum standard length and Fulton's condition index -K. A parasitological exam was performed on fish samples examining accurately the coelomic cavity and muscle by visual and stereoscopic inspection.
For visual and stereoscopic inspection, the fish were dissected by making an incision along the ventral line in a caudocranial direction. Subsequently, a chloro-peptic digestion of viscera and muscles was carried out according to the protocol reported by Cammilleri et al.(2016). All the nematode larvae collected were washed in saline solution, fixed in 70 % ethanol and cleared with glycerol for morphological identification by light microscopy Leica DM 2000 (Wetzlar, Germany), following the taxonomic keys (Berland, 1961).

Molecular analysis
All the larvae were submitted to molecular identification at species level by PCR-based restriction fragment length polymorphism (PCR-RFLP) analysis of the rDNA comprising the internal transcribed spacers ITS (ITS-1, 5.8S gene, and ITS-2) region.
Furthermore, a PCR amplification and sequencing of the ITS region was carried out. The larvae were washed, fragmented with a scalpel and frozen at -20 °C for 24 hours. Genomic DNA extraction were conducted by special kits based on the use of affinity columns (Omega Bio-tek Inc., Norcross, USA), according to the manufacturer's instructions.
The nuclear rDNA containing ITS region was amplified using NC5 (5' (2000). The ITS gene amplification products were also purified with Illustra GFX PCR DNA (GE Healthcare, Boston, Massachusetts) and Gel Band Purification kit (GE Healthcare) following the manufacturer's instructions for sequencing analysis. The purified products were sent to Macrogen company (Amsterdam, Holland) for Sanger sequencing.

Data analysis
Infestation parameters such as prevalence and mean intensity were examined using the software Quantitative Parasitology 3.0. The raw sequence data were manually analyzed for quality assessment and compared with previously characterized ITS sequences of Anisakidae published for identification by using the Basic Local Alignment Search Tool (BLAST) via GenBank.

Submission of sequence data to databases
Nucleotide sequence data reported in this paper are available in the GenBank™ databases under the accession number: MH197040 (Anisakis pegreffii ITS region).

RESULTS
All the European sea bass samples were of commercial size with a mean weight and standard deviation (SD) of 402.78 ± 49.72. The weight, maximum standard length, Fulton's condition index and infestation parameters of the fish samples examined are shown in Table 2. A Fulton's condition -K index above 2 was found in 12 samples from Sicilian and Greek farms. Five samples came from Greek farms showed a K index below 1. Two nematode larvae were found in an European sea bass sample from farms of Greece after visual inspection, giving a prevalence of infestation of 0.7%, a mean abundance of 0.01 and a mean intensity of 1. The larvae were located under the visceral serosa of intestine. No additional larvae were found after chloro-peptic digestion. No co-infestation with other parasites was found. The weight of the affected fish was lower than the mean weight, detecting a K index of 0.83. The morphological analysis allowed to identify parasites as L3 Anisakis larvae belonging to the morphotype I (sensu Berland 1961). The amplification of ITS region produced a single band of ≈1000 bp for all the specimens. The PCR-RFLP analysis of ITS region, obtained with HinfI restriction enzyme produced one pattern with three strong bands at 370, 300 and 250 bp corresponding to Anisakis pegreffii. The digestion with HhaI produced one pattern with two bands (550-430 bp) (Figure 2). The PCR-RFLP results were confirmed by sequence analysis. The alignment of rDNA ITS sequences with the sequences deposited in GenBank confirmed the results obtained by PCR-RFLP analysis. The alignment with BLAST showed a 99% of identity and 100% query cover with sequence of A. pegreffii from Mediterranean Sea (KY426257.1).

DISCUSSION
To our knowledge this is the first report on the presence of Anisakis larvae in farmed European sea bass. Although digestion method is considered a valid method to recover larvae from viscera and muscle tissue, no additional parasites were found. Our findings seemingly contrast previous studies that confirmed the absence of Anisakis nematodes in 259 farmed European sea bass from Southeast Spain (Peñalver et al., 2010). No Anisakis larvae were found even in 1040 European sea bass samples from North Italy (Menconi et al., 2017)  Based on what is reported on the National Aquaculture Sector Overview of the Food and Agriculture Organization of the United Nations (FAO), Greek aquaculture is dominated by the farming of marine finfish in offshore cages, specifically of gilthead sea bream and European sea bass (FAO, 2005).
Therefore, it is possible that this that this sample infected by Anisakis was reared in offshore opennet cages. This farming practices could allow the exposure of fish to the crustacean intermediate host (Peñalver et al., 2010). Furthermore, the European sea bass sample infected by Anisakis showed a low Fulton's K index (0.83). Given that the Fulton's K condition index is considered a good indicator of the general welfare of the fish (Lambert and Dutil, 1997), we assume particular stressed condition that lead the fish to feed on infected crustaceans and small fish entering the cage.
Most of the studies on the presence of anisakid parasites in farmed fish have focused on salmonids.
Several authors have demonstrated the absence of anisakids in farmed Atlantic salmon (Salmo salar) (Angot and Brasseur, 1993;Lunestad, 2003). Marty (2008) reported the presence of an anisakid larva in a farmed Atlantic salmon, although molecular identification was not carried out. The presence of these parasites was verified even in runts of farmed Atlantic salmon (Mo et al., 2013). Based on the prevalence values reported in literature (Bernardi et al., 2011), the relative risk of anisakids in European sea bass is estimated to be 136 times greater in wild forms than in farmed forms.
At present, there are few data on the prevalence of anisakid parasites in farmed European sea bass (Menconi et al., 2017;Mladineo et al., 2010;Peñalver et al., 2010). This could be because these fish are not usually consumed raw and consequently are not considered a potential risk in terms of zoonosis. Nevertheless, several studies that point out the allergic potential of Anisakis spp. in clinical reports and the presence of thermally stable allergens in processed fish products (Faeste et al., 2015;Audicana and Kennedy, 2008) encourage to increase the monitoring data on farmed fish of Mediterranean, in order to clarify the effects of different farming practices on the prevalence of Anisakidae nematodes and fulfil the EFSA Panel on Biological Hazards' requests (EFSA, 2010). In conclusion, we showed for the first time the presence of Anisakidae nematodes in farmed European sea bass. Therefore, our findings should be considered in future risk analyses on the fish species farmed in Mediterranean. However, due to the low prevalence found in this work, the risk of exposure to anisakid parasites in farmed fish remain very low.