Fishing area and fish size as risk factors of Anisakis infection in sardines (Sardina pilchardus) from Iberian waters, southwestern Europe Dolores Molina-Fernández, David Malagón, Magdalena Gómez-Mateos, Rocío Benítez, Joaquina Martín-Sánchez, Francisco Javier Adroher ⁎ Departamento de Parasitología, Facultad de Farmacia, Universidad de Granada, 18071 Granada, Spain abstract article info Article history: Received 14 September 2014 Received in revised form 23 December 2014 Accepted 20 February 2015 Available online 28 February 2015 Keywords: Anisakiasis Anisakis simplex s.l. Hybrids Epidemiology Infection risk factors The sardine (Sardina pilchardus) is a fish commonly consumed and appreciated in many countries, although they are more likely to be eaten fresh in western Mediterranean countries such as Spain, Portugal, France or Italy. A mo- lecular epidemiological survey of sardines from 5 fishing areas of the Spanish Mediterranean (Málaga, southern Spain) and Atlantic coasts (southern: Cádiz and Isla Cristina; northern: A Coruña and Ondarroa) was carried out to determine the presence of Anisakis spp. larvae. The highest prevalence of these larvae was observed in fish from A Coruña (28.3%), followed by Ondarroa (5%) and Cádiz (2.5%). No Anisakis larvae were found in fish from Málaga and Isla Cristina. Three Anisakis genotypes were identified: Anisakis simplex sensu stricto, Anisakis pegreffii and a hybrid genotype between these two species. A. pegreffii was the most prevalent species in A Coruña (71% of larvae). Only three Anisakis larvae (9% collected larvae) were located in the musculature of sardines: two were identified as A. pegreffii while the other was a hybrid genotype. Sardine infection was associated with fishing area and fish length/weight (length and weight were strongly correlated; Pearson's correlation 0.82; p b 0.001). Risk factor multivariate analysis showed that the risk of infection increases 1.6 times for every additional cm in the length of the sardines from the same fishing area. Comparison of fish of equal length showed that in sardines from A Coruña the risk of parasitization is 11.5 times higher than in those from other fishing areas. Although the risk of infection by Anisakis through consumption of sardines is generally low due to the low epidemiological parameter values (prevalence 10%, mean intensity 1.7 (range 1–5) and mean abundance 0.17), as larger fish are more heavily parasitized, there is an increased risk of infection by Anisakis through consumption of large sardines which are raw or have undergone insufficient treatment (undercooked, smoked, marinated, salted, pickled, freezing,…). © 2015 Elsevier B.V. All rights reserved. 1. Introduction The sardine (Sardina pilchardus) is a littoral fish which feeds mainly on planktonic crustaceans, appendicularians, diatoms and other organ- isms (Costalago and Palomera, 2014). This fish is marketed fresh, frozen or canned. It is also consumed dried or salted and smoked or marinated and can be pan-fried, broiled and microwaved. Sardines can harbor parasites such as Anisakis, which are transmitted to humans. Anisakis spp. are nematodes which can parasitize a wide range of marine animals. The third larval stage (L3) of this parasite is the etiolog- ical agent of human anisakiasis, a disease that causes gastric and intes- tinal illness and/or allergic reactions. The larvae of Anisakis, dead or alive, are also considered to cause food allergy, although this is currently under discussion (Audicana and Kennedy, 2008; Daschner et al., 2012). Reports of cases of anisakiasis are increasing globally. The majority of cases have been reported in Japan, where consumption of raw fish is extremely common. The life cycle of Anisakis is complex and involves a large number of host species. Larvae of Anisakis have been reported in numerous inver- tebrate hosts, mainly crustaceans, which can act as intermediate hosts. L3 of this parasite have been found in a wide range of fish and cephalo- pods, which are intermediate/paratenic hosts. Anisakis parasitization has been reported in more than 200 fish and 25 cephalopod species (Abollo et al., 2001; Klimpel et al., 2004). Cetaceans (final hosts) harbor the adult stage of this nematode. Humans can become accidental hosts, by eating raw, marinated or undercooked fish containing the L3 of these parasites that have not been inactivated during preparatory procedures. Anisakis type I larvae have been categorized into six species: Anisakis simplex sensu stricto, Anisakis pegreffii, Anisakis berlandi, Anisakis typica, Anisakis ziphidarum and Anisakis nascetti (Mattiucci and Nascetti, 2008; Mattiucci et al., 2009, 2014). Although several species have been found to parasitize fish and cephalopods in Japan, human anisakiasis is caused almost exclusively by A. simplex s.s. larvae in this country (Arizono et al., 2012; Umehara et al., 2007). Eleven clinical cases attributed to A. pegreffii have been reported in Italy, where this species is the dominant in Italian waters whereas none due to A. simplex s.s. have been described to date in this area (D'Amelio et al., International Journal of Food Microbiology 203 (2015) 27–34 ⁎ Corresponding author. E-mail address: fadroher@ugr.es (F.J. Adroher). http://dx.doi.org/10.1016/j.ijfoodmicro.2015.02.024 0168-1605/© 2015 Elsevier B.V. All rights reserved. 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