Advertisement

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
Article
  • 194 Downloads

Abstract

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.

Keywords

Anisakids UV press method Enzymatic digestion Food safety Method validation 

Notes

Compliance with Ethical Standards

Funding

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)

References

  1. Adams AM, Murrell KD, Cross JH (1997) Parasites of fish and risks to public health. Rev Sci Tech (Int Off Epizoot) 16:652–660CrossRefGoogle Scholar
  2. Anderson RC (1992) Nematode parasites of vertebrates: their development and transmission. CAB International, CambridgeGoogle Scholar
  3. Angelucci G, Meloni M, Merella P, Sardu F, Madeddu S, Marrosu R, Petza F, Salati F (2011) Prevalence of Anisakis spp. and Hysterothylacium spp. larvae in teleosts and cephalopods sampled from waters off Sardinia. J Food Prot 74:1769–1775CrossRefGoogle Scholar
  4. Alonso-Gomez A, Moreno-Ancillo A, Lopez-Serrano MC, Suarez-de-Parga JM, Daschner A, Caballero MT et al (2004) Anisakis simplex only provokes allergic symptoms when the worm parasitises the gastrointestinal tract. Parasitol Res 93:378–384Google Scholar
  5. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic Local Alignment Search Tool. J Mol Biol 215:403–410CrossRefGoogle Scholar
  6. Armani A, Tinacci L, Xiong X, Titarenko E, Guidi A, Castigliego L (2014) Development of a simple and cost-effective bead-milling method for DNA extraction from fish muscles. Food Anal Methods 7:946–955CrossRefGoogle Scholar
  7. Audicana MT, Ansotegui IJ, Corres LF, Kennedy MW (2002) Anisakis simplex: dangerous—dead and alive? Trends Parasitol 18:20–25CrossRefGoogle Scholar
  8. Bernardi C, Gustinelli A, Fioravanti ML, Caffara M, Mattiucci S, Cattaneo P (2011) Prevalence and mean intensity of Anisakis simplex (sensu stricto) in European sea bass (Dicentrarchus labrax) from Northeast Atlantic Ocean. Int J Food Microbiol 148:55–59CrossRefGoogle Scholar
  9. Borges JN, Cuhna LFG, Santos HLC, Monteiro-Neto C, Santos CP (2012) Morphological and molecular diagnosis of anisakid nematode larvae from cutlassifish (Trichiurus lepturus) off the Coast of Rio de Janeiro, Brazil. PLoS One 7:e40447CrossRefGoogle Scholar
  10. Bush AO, Lafferty KD, Lotz JM, Shostak AW (1997) Parasitology meets ecology on its own terms: Margolis et al. revisited. J Parasitol 83:575–583CrossRefGoogle Scholar
  11. Butt AA, Aldridge KE, Sander CV (2004) Infections related to the ingestion of seafood. Part II: parasitic infections and food safety. Lancet Infect Dis 4:294–300CrossRefGoogle Scholar
  12. Cavallero S, Magnabosco C, Civettini M, Boffo L, Mingarelli G, Buratti P, Giovanardi O, Fortuna CM, Arcangeli G (2015) Survey of Anisakis sp. and Hysterothylacium sp. in sardines and anchovies from the North Adriatic Sea. Int J Food Microbiol 200:18–21CrossRefGoogle Scholar
  13. Chai JY, Murrell KD, Lymbery AJ (2005) Fish-borne parasitic zoonoses: status and issues. Int J Parasitol 35:1233–1254CrossRefGoogle Scholar
  14. Cipriani P, Smaldone G, Acerra V, D’Angelo L, Anastasio A, Bellisario B, Palma G, Nascetti G, Mattiucci S (2015) Genetic identification and distribution of the parasitic larvae of Anisakis pegreffii and Anisakis simplex (ss) in European hake Merluccius merluccius from the Tyrrhenian Sea and Spanish Atlantic Coast: implications for food safety. Int J Food Microbiol 198:1–8CrossRefGoogle Scholar
  15. Circular Letter VS8/C790/94 of the Lombardy RegionGoogle Scholar
  16. Circular (1997) n. 1 of Liguria RegionGoogle Scholar
  17. Codex Alimentarius Commission (1969) Codex sampling plans for prepackaged foods. CODEX STAN 233–1969. Available at: http://down.40777.cn/stardard/10/CODEX%20STAN%20233-1969%20CODEX%20SAMPLING%20PLANS%20FOR%20PREPACKAGED%20FOODS%20%28AQL%206.5%29.pdf Accessed 14/05/2015
  18. Codex Alimentarius Commission (1971) Report of the eighth session of the joint FAO/WHO Codex Alimentarius Commission: recommended international standard for quick frozen filet of cod and haddock. CAC/RS-50-1971. Available at: http://www.fao.org/docrep/meeting/005/c0531e/C0531E09.htm Accessed 16/02/2015
  19. Codex Alimentarius Commission (1989) Codex standard for quick frozen blocks of fish fillet, minced fish flesh and mixtures of fillets and minced fish flesh. CODEX STAN 165–1989. Available at: http://www.codexalimentarius.org/standards/list-of-standards/ Accessed 09/06/2015
  20. Codex Alimentarius Commission (2004) Standard for salted Atlantic herring and salted sprat CODEX STAN 244–2004 Available at: http://www.codexalimentarius.org/standards/list-of-standards/ Accessed 09/06/2015
  21. Commission Decision (EEC) (1993) No 140 Laying down the detailed rules relating to the visual inspection (or the purpose of detecting parasites in fishery products). OJEC L56:42Google Scholar
  22. Commission Regulation (EC) (2005) No 2074/2005 Laying down implementing measures for certain products under regulation (EC) No. 853/2004 of the European parliament and of the council and for the organisation of official control under regulation (EC) No. 854/2004 of the European parliament and of the council and regulation (EC) No. 882/2004 of the European parliament and of the council, derogating from regulation (EC) No. 852/2004 of the European parliament and of the council and amending regulations (EC) No. 853/2004 and (EC) No. 854/2004. OJEU L338:27–59Google Scholar
  23. Commission Regulation (EC) (2005) No 2075/2005 Laying down specific rules on official controls for Trichinella in meat. OJEU L338:60Google Scholar
  24. Commission Regulation (EU) (2011) No 1276/2011 amending Annex III to regulation (EC) No 853/2004 of the European parliament and of the council as regards the treatment to kill viable parasites in fishery products for human consumption. OJEU L327:39–41Google Scholar
  25. Council Regulation (EC) (1996) No 2406/96 Laying down common marketing standards for certain fishery products. OJEC L334:1–15Google Scholar
  26. Daschner A, Alonso-Gomez A, Cabanas R, Suarez-de-Parga JM, Lopez-Serrano MC (2000) Gastroallergic anisakiasis: borderline between food allergy and parasitic disease: clinical and allergologic evaluation of 20 patients with confirmed acute parasitism by Anisakis simplex. J Allergy Clin Immunol 105:176–181CrossRefGoogle Scholar
  27. D’Amico P, Malandra R, Costanzo F, Castigliego L, Guidi A, Gianfaldoni D, Armani A (2014) Evolution of the Anisakis risk management in the European and Italian context. Food Res Int 64:348–362CrossRefGoogle Scholar
  28. Dominguez-Ortega J, Alonso-Llamazares A, Rodriguez L, Chamorro M, Robledo T, Bartolome JM et al (2001) Anaphylaxis due to hypersensitivity to Anisakis simplex. Int Arch Allergy Immunol 125:86–88Google Scholar
  29. EFSA (2010) Scientific opinion on risk assessment of parasites in fishery products. EFSA Journal 8:1543 Available at: http://www.efsa.europa.eu/it/search/doc/1543.pdf Accessed 10/05/2015
  30. Fagerholm HP (1991) Systematic implications of male caudal morphology in ascaridoid nematode parasites. Syst Parasitol 19:215–229CrossRefGoogle Scholar
  31. Fraulo P, Morena C, Costa A (2014) Recovery of anisakid larvae by means of chloro-peptic digestion and proposal of the method for the official control. Acta Parasitol 59:629–634CrossRefGoogle Scholar
  32. Huang W (1990) Methods for detecting anisakid larvae in marine fish. Possibilities of application to the inspection of fish sold in the Paris Region. Recueil de Médecine Vétérinaire 166:895–900Google Scholar
  33. ISMEA (2013) Il settore ittico in Italia. Check-up – 2013. Available at: http://www.ismea.it/flex/cm/pages/ServeBLOB.php/L/IT/IDPagina/8845 Accessed 20/04/2015
  34. Karl H, Leinemann M (1993) A fast and quantitative detection method for nematodes in fish fillets and fishery products. Arch Leb 44:124–125Google Scholar
  35. Levsen A, Lunestad BT, Berland B (2005) Low detection efficiency of candling as a commonly recommended inspection method for nematode larvae in the flesh of pelagic fish. J Food Prot 68:828–832Google Scholar
  36. Lima dos Santos CAM, Howgate P (2011) Fishborne zoonotic parasites and aquaculture: a review. Aquaculture 318:253–261CrossRefGoogle Scholar
  37. Llarena-Reino M, González ÁF, Vello C, Outeiriño L, Pascual S (2012) The accuracy of visual inspection for preventing risk of Anisakis spp. infection in unprocessed fish. Food Control 23:54–58CrossRefGoogle Scholar
  38. Llarena-Reino M, Piñeiro C, Antonio J, Outeriño L, Vello C, González AF, Pascual S (2013a) Optimization of the pepsin digestion method for anisakids inspection in the fishing industry. Vet Parasitol 191:276–293CrossRefGoogle Scholar
  39. Llarena-Reino M, Abollo E, Pascual S (2013b) A scoring system approach for the parasite predictive assessment of fish lots: a proof of concept with anisakids. Foodborne Pathog Dis 10:1067–1074CrossRefGoogle Scholar
  40. Lymbery AJ, Cheah FY (2007) Anisakid nematodes and anisakiasis. In: Murrell KD, Fried B (eds) Food-borne parasitic zoonoses. Springer, New York, pp 185–206CrossRefGoogle Scholar
  41. Mattiucci S, Nascetti G (2008) Advances and trends in the molecular systematics of anisakid nematodes, with implications for their evolutionary ecology and host-parasite co-evolutionary processes. Adv Parasitol 66:47–148CrossRefGoogle Scholar
  42. Mattiucci S, Fazii P, De Rosa A, Paoletti M, Megna AS, Glielmo A, De Angelis M, Costa A, Meucci C, Calvaruso V, Sorrentini I, Palma G, Bruschi F, Nascetti G (2013) Anisakiasis and gastroallergic reactions associated with Anisakis pegreffii infection, Italy. Emerg Infect Dis 19:496–499CrossRefGoogle Scholar
  43. Messing J (1983) New M13 vectors for cloning. Methods Enzymol 101:20–78CrossRefGoogle Scholar
  44. Mladineo I, Poljak V (2013) Ecology and genetic structure of zoonotic Anisakis spp. from adriatic commercial fish species. Appl Environ Microbiol 80:1281–1290CrossRefGoogle Scholar
  45. Murata R, Suzuki J, Sadamasu K, Kai A (2011) Morphological and molecular characterization of Anisakis larvae (Nematoda: Anisakidae) in Beryx splendens from Japanese waters. Parasitol Int 60:193–198Google Scholar
  46. Nadler AS, Hudspeth DSS (2000) Phylogeny of the Ascaridoidea (Nematoda: Ascaridida) based on three genes and morphology: hypotheses of structural and sequence evolution. J Parasitol 86:380–393CrossRefGoogle Scholar
  47. Nieuwenhuizen N, Lopata AL, Jeebhay MF, De’Broski RH, Robins TG, Brombacher F (2006) Exposure to the fish parasite Anisakis causes allergic airway hyperreactivity and dermatitis. J Allergy Clin Immunol 117:1098–1105Google Scholar
  48. Nieuwenhuizen NE, Lopata AL (2013) Anisakis—a food-borne parasite that triggers allergic host defences. Int J Parasitol 43:1047–1057CrossRefGoogle Scholar
  49. Pardo-Gandarillas MC, Lohrmann KB, Valdivia AL, Ibáñez CM (2009) First record of parasites of Dosidicus gigas (d’Orbigny, 1835) (Cephalopoda: Ommastrephidae) from the Humboldt current system off Chile. Rev Biol Mar Oceanogr 44:397–408CrossRefGoogle Scholar
  50. Piras MC, Tedde T, Garippa G, Virgilio S, Sanna D, Farjallah S, Merella P (2014) Molecular and epidemiological data on Anisakis spp. (Nematoda: Anisakidae) in commercial fish caught off northern Sardinia (western Mediterranean Sea). Vet Parasitol 203:237–240CrossRefGoogle Scholar
  51. Regulation (EC) (2002) No 178 of the European parliament and of the council of 28 January 2002 laying down the general principles and requirements of food law, establishing the European food safety authority and laying down procedures in matters of food safety. OJEC L31:1–24Google Scholar
  52. Regulation (EC) (2004) No 852 of the European parliament and of the council of 29 April 2004 on the hygiene of foodstuffs. OJEU L139:1Google Scholar
  53. Regulation (EC) (2004) No 853 of the European parliament and of the council of 29 April 2004 laying down specific hygiene rules for on the hygiene of foodstuffs. OJEU L139:55Google Scholar
  54. Rello FJ, Adroher FJ, Benitez R, Valero A (2009) The fishing area as a possible indicator of the infection by anisakids in anchovies (Engraulis encrasicolus) from southwestern Europe. Int J Food Microbiol 129:277–281CrossRefGoogle Scholar
  55. Rózsa L, Reiczigel J, Majoros G (2000) Quantifying parasites in samples of hosts. J Parasitol 86:228–232CrossRefGoogle Scholar
  56. Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425Google Scholar
  57. SANCO (2013) 10137/2013-rev1 guidance on the term ‘obviously contaminated’ in relation to parasites in fishery products. Working document SANCO/10137/2013-rev1. Available at: http://multimedia.food.gov.uk/multimedia/pdfs/obviouslycontaminated. Accessed 16/04/2015
  58. Shamsi S, Eisenbarth A, Saptarshi S, Beveridge I, Gasser RB, Lopata AL (2011) Occurrence and abundance of anisakid nematode larvae in five species of fish from southern Australian waters. Parasitol Res 108:927–934CrossRefGoogle Scholar
  59. Shamsi S, Gasser R, Beveridge I (2013) Description and genetic characterisation of Hysterothylacium (Nematoda: Raphidascarididae) larvae parasitic in Australian marine fishes. Parasitol Int 62:320–328CrossRefGoogle Scholar
  60. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30:2725–2729CrossRefGoogle Scholar
  61. Yagi K, Nagasawa K, Ishikura H, Nagasawa A, Sato N, Kikuchi K, Ishikura H (1996) Female worm Hysterothylacium aduncum excreted from human: a case report. Jpn J Parasitol 45:12–23Google Scholar
  62. Regulation (EU) (2011) No 1169 of the European parliament and of the council of 25 October 2011 on the provision of food information to consumers, amending regulations (EC) No 1924/2006 and (EC) No 1925/2006 of the European parliament and of the council, and repealing commission directive 87/250/EEC, council directive 90/496/EEC, commission directive 1999/10/EC, directive 2000/13/EC of the European parliament and of the council, commission directives 2002/67/EC and 2008/5/EC and commission regulation (EC) No 608/2004. OJEU L304:18Google Scholar

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

Personalised recommendations