Food Analytical Methods

, Volume 9, Issue 5, pp 1418–1427 | Cite as

Assessment of a Sampling Plan Based on Visual Inspection for the Detection of Anisakid Larvae in Fresh Anchovies (Engraulis encrasicolus). A First Step Towards Official Validation?

  • L. Guardone
  • R. Malandra
  • F. Costanzo
  • L. Castigliego
  • L. Tinacci
  • D. Gianfaldoni
  • A. Guidi
  • A. ArmaniEmail author


The presence of anisakid larvae in fish is a public health issue, and effective risk management procedures are needed to avoid that heavily infected products reach the market. Currently, an official sampling plan for fresh fish defining sample size, inspection methods, and criteria to accept or reject the merchandise is lacking at the European and Italian level. In this study, we compared the visual inspection proposed by the sampling plan of the Lombardy Region (Italy) to the UV press method and to an optimized digestion procedure with the aim to assess its ability in detecting visible parasites. Thirty-one batches of Engraulis encrasicolus, each composed of ∼30 specimens, were collected and subsequently analyzed with the three techniques. The mean abundance (MA) was calculated after each procedure and compared on the basis of a threshold value. The results showed that the visual inspection performed similarly to the digestion method, with a sensitivity of 93 %, a specificity of 100 %, and an accuracy of 97 %. Overall, the comparison showed that, in the proposed sampling plan, the visual inspection is effective in rejecting unmarketable anchovies and in preventing the commercialization of unsafe products. This method is simple, less demanding than digestion in terms of time and equipment, and thus suitable as a standardized procedure to be routinely applied by food business operators. The hazard characterization, performed by sequencing the mtDNA cox2 gene, has identified the visible larvae as Anisakis pegreffii in 98 % of the cases, highlighting the zoonotic potential of the parasites found and the need for preventive measures.


Anisakids UV press method Enzymatic digestion Food safety Method validation 


Compliance with Ethical Standards


The research was performed with funds granted from the University of Pisa.

Conflict of Interest

Lisa Guardone declares that she has no conflict of interest. Renato Malandra declares that he has no conflict of interest. Francesco Costanzo declares that he has no conflict of interest. Lorenzo Castigliego declares that he has no conflict of interest. Lara Tinacci declares that she has no conflict of interest. Daniela Gianfaldoni declares that she has no conflict of interest. Alessandra Guidi declares that she has no conflict of interest. Andrea Armani declares that he has no conflict of interest. This article does not contain any study with human or animal subjects. In particular, the fish included in the present study were intended for human consumption and they were bought at the wholesale market of Milan.

Supplementary material

12161_2015_316_MOESM1_ESM.tiff (270 kb)
Fig. 1SM Neighbour-joining (NJ) tree obtained using 622 mtDNA cox2 gene sequences (576 bp) of the Anisakis spp. larvae found in this study (613) and the reference sequences (9) retrieved from Genbank. Bootstrap values >70 %, obtained from 2000 replications using the Kimura 2-parameter genetic distance are shown in the tree. (TIFF 269 kb)
12161_2015_316_Fig2_ESM.gif (80 kb)
High Resolution (GIF 80 kb)
12161_2015_316_MOESM2_ESM.doc (60 kb)
Table 1SM Sampling for the visual inspection to detect Anisakis larvae in fish batches, according to the Circular Letter VS8/C790/94 of the Lombardy region (adapted and published in D’Amico et al. 2014). (DOC 60 kb)
12161_2015_316_MOESM3_ESM.doc (75 kb)
Table 2SM Molecular results of the BLAST analysis performed on a fragment of ∼582 bp of the cox2 gene. (DOC 75 kb)


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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • L. Guardone
    • 1
  • R. Malandra
    • 2
  • F. Costanzo
    • 1
  • L. Castigliego
    • 1
  • L. Tinacci
    • 1
  • D. Gianfaldoni
    • 1
  • A. Guidi
    • 1
  • A. Armani
    • 1
    Email author
  1. 1.FishLab, Department of Veterinary SciencesUniversity of PisaPisaItaly
  2. 2.Wholesale Fish Market of MilanASL of MilanMilanItaly

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