Anisakis spp. larvae in different kinds of ready to eat products made of anchovies (Engraulis encrasicolus) sold in Italian supermarkets

In this study the occurrence of visible anisakid larvae in semi-preserved anchovy products sold on the Italian market was investigated. Totally, 107 ready to eat products (33 salted-ripened, 49 in oil and 25 marinated) were sampled. Each sample was digested, then the digested material was observed under natural and UV light. Parasites were counted, collected and microscopically identified to genus level. A representative subset was molecularly identified using the cox2 gene. At least one visible Anisakis sp. larva was found in 54.2% of the total 107 products analysed and totally 1283 dead larvae were collected. Anisakis sp. larvae were found in all the 33 salted products and 1139 (88.8%) larvae were collected, with a range of 1â105 parasites per product. Larval density per gram was 0.13. Anisakis sp. larvae were found in 49.0% of the products in oil and 143 (11.1%) larvae were isolated, with a range of 0â28 and a density of 0.03. Only 1 larva was found in the 25 marinated products (4.0%, density 0.00). A highly significant difference between all the product categories in respect of number of larvae per product, frequency of products contaminated by at least one larva and larval density per gram was found. Within the subset of larvae molecularly analysed (n = 122), 92 (75.4%) were identified as A. pegreffii and 30 (24.6%) as A. simplex. This study showed that semi-preserved anchovy products heavily contaminated with Anisakis spp. larvae reach the market. Beyond the negligible risk for anisakidosis, the presence of dead visible parasites may cause immediate rejection in consumers. In addition, the potential risk related to allergic reactions in sensitized individuals needs to be further assessed. In order to avoid commercialization of obviously contaminated products, fresh anchoviesâ batches intended for the production of such products should be accurately selected by the processing industry applying inspection methods

Buccal swabs for long-term DNA storage in conservation genetics of fish: One-and-a-half-year analysis timeframe

Conservation genetic research is essential for the management and recovery of endangered taxa. However, the invasive collection of biological material for DNA analysis is controversial. From an ethical perspective, non-destructive sampling methods leave the aquatic specimen alive and less invasive procedures minimize stress on the animals. DNA can be obtained from fish using minimally invasive techniques such as buccal swabs. Here we evaluated the performance of buccal swabs for long-term storage of DNA obtained from brown trout (Salmo trutta). The buccal swabs were stored at room temperature and cut into pieces, one part of which was used for extraction of an aliquot and the others were stored as a “biobank” of biological material. The elapsed time from sampling to molecular analysis was one and half year. The amplification of three different DNA targets was tested to assess the effectiveness of the extraction: mitochondrial DNA (the D-LOOP region), nuclear DNA (the LDH gene) and microsatellite DNA at multiple loci. The results showed high quantification (mean value: 281.84±72.4 ng/μL), indicating that DNA could be effectively extracted from the buccal swabs. Our study results suggest that buccal swabs for long-term storage of DNA at room temperature are promising for use in field conservation studies

The gut microbiota of farmed and wild brook trout (Salvelinus fontinalis): evaluation of feed-related differences using 16S rRNA gene metabarcoding

The gut microbiota has become a topic of increasing importance in various fields, including aquaculture. Several fish species have been the subject of investigations concerning the intestinal microbiota, comparing different variables including the intestine portions, the environment and diet. In this study, the microbiota of farmed and wild brook trout (Salvelinus fontinalis) was analysed, considering separately wall and content of the medial portion of the intestine. A total of 66 fish (age class 2+) were sampled, of which 46 wild and 20 farmed brook trout, along two different years. Microbiota data were obtained using a 16S metabarcoding approach by analysing the V3-V4 hypervariable regions of the 16S rRNA. Data showed that the core microbiota of these species is represented by Proteobacteria (Alpha- and Gammaproteobacteria), Actinobacteria, Firmicutes (Bacilli and Clostridia) and, only for farmed animals, Fusobacteria. The latter taxon is likely related to the fishmeal-based diet administered to farmed brook trout. Indeed, alpha and beta diversity analysis showed differences between wild and farmed fish. Finally, statistically significant differences in the microbiota composition were observed between intestinal wall and content in wild fish, while no differences were detected in reared animals

Immune profiling of rainbow trout (Oncorhynchus mykiss) exposed to Lactococcus garvieae: Evidence in asymptomatic versus symptomatic or vaccinated fish

Lactococcosis, caused by the Gram-positive bacterium Lactococcus garvieae, is a major concern in rainbow trout (Oncorhynchus mykiss) farms, which are regularly affected by outbreaks especially during the summer/fall months. In these farms, unvaccinated healthy and symptomatic fish can coexist with vaccinated fish. In the present study, innate (leukogram, serum lysozyme activity, peroxidase activity, antiprotease activity, bactericidal activity, total IgM and total proteins), and specific immune parameters (serum antibodies to L. garvieae) were assessed in unvaccinated adult rainbow trout naturally exposed to the pathogen, with or without evidence of clinical signs, or subjected to vaccination. Blood was drawn from all three groups, and blood smears were prepared. Bacteria were found in the blood smears of 70% of the symptomatic fish but not in any of the asymptomatic fish. Symptomatic fish showed lower blood lymphocytes and higher thrombocytes than asymptomatic fish (p ≤.05). Serum lysozyme and bactericidal activity did not vary substantially among groups; however, serum antiprotease and peroxidase activity were significantly lower in the unvaccinated symptomatic group than in the unvaccinated and vaccinated asymptomatic groups (p ≤.05). Serum total proteins and total immunoglobulin (IgM) levels in vaccinated asymptomatic rainbow trout were significantly higher than in unvaccinated asymptomatic and symptomatic groups (p ≤.05). Similarly, vaccinated asymptomatic fish produced more specific IgM against L. garvieae than unvaccinated asymptomatic and symptomatic fish (p ≤.05). This preliminary study provides basic knowledge on the immunological relationship occurring between the rainbow trout and L. garvieae, potentially predicting health outcomes. The approach we proposed could facilitate infield diagnostics, and several non-specific immunological markers could serve as reliable indicators of the trout's innate ability to fight infection

The association of Lactococcus petauri with lactococcosis is older than expected

Lactococcosis is a globally prevalent infectious disease that has a significant economic and sanitary impact on the rainbow trout industry. Lactococcus garvieae has traditionally been considered the only species implicated in the etiology of this disease, but Lactococcus petauri, a new species, has recently been implicated as another etiological agent. Both species cannot be distinguished by routine methods commonly used in diagnostic laboratories, resulting in their misidentification. In the present study, the identification of 48 isolates initially identified as L. garvieae was studied by determining their in-silico DNA–DNA hybridization (dDDH) and average nucleotide identity (ANI) values using pairwise comparisons of their whole genome sequences and the genomes of the type strains of L. garvieae and L. petauri. The genome sequences of 37 isolates from countries in which lactococcosis can be considered endemic (Spain, Italy, Türkiye, and Greece) were obtained in this study, and the genomes of 11 isolates were retrieved from the GenBank database. Isolates from Italy, Singapore, Japan, South Korea, India, one Turkish isolate from 2013 and two Spanish isolates recovered in 1992 and 1996 were confirmed as L. garvieae. The remaining isolates from Spain and Türkiye, as well as those from Portugal, Israel, USA, and Greece were identified as L. petauri. Overall, 60.4% of isolates previously identified as L. garvieae were found to be L. petauri. These results confirm the implication of both species in the etiology of lactococcosis and suggest that L. petauri plays a significant role in the epidemiology of this disease. Some of the isolates identified as L. petauri in the present study were isolated three decades ago, indicating that its association with lactococcosis is older than might be expected from the recent descriptions. The commercial Rapid ID32 Strep system was unable to discriminate between L. garvieae and L. petauri. However, both species exhibited some biochemical differences that might serve as phenotypic markers for their presumptive recognition. Consequently, isolates that hydrolyze hippurate and produce acid from sucrose and tagatose could be presumptively recognized as L. petauri, while those that fail these tests could be identified as L. garvieae. The results of this work indicate that great attention should be given to L. petauri in the epidemiology of lactococcosis