Estimation of the number of Anisakis larvae in commercial fish using a descriptive model based on real-time PCR

BACKGROUND: Seafood parasitation by Anisakis (Anisakidae) larvae has been reported in most of the oceans and seas worldwide.The presence of these nematodes in commonly consumed fish represents a potential hazard for consumers as they can provoke gastrointestinal symptoms and allergic reactions. In the present work, the capacity of a SYBR Green qPCR protocol to quantify Anisakis larvae in commercial fish was evaluated using experimentally spiked samples with different numbers (0-50) of A. simplex third-stage larvae (L3). To verify the agreement of the obtained results, 25 naturally infected fish specimens of Atlantic blue whiting underwent a parallel visual inspection. RESULTS: The logarithmic behavior of the Cq data obtained from the experimentally spiked samples allowed the development of a descriptive mathematical model that correlates the Cq value with the number of Anisakis larvae (R2 = 0.9908, CV = 2.37%). In the commercial blue whiting specimens there was a high correlation between the results of the molecular technique and the visual inspection (R2 = 0.9912); the Bland-Altman analysis showed that 94% of the differences were within the limits of agreement (−4.98 and 6.68), indicating the reliability of the descriptive mathematical model based on the SYBR Green qPCR technique. CONCLUSION: The descriptive function presented based on the SYBR Green qPCR assay is promising as a sensitive and accurate tool for measuring the Anisakis larval load in commercial fish, with a potential application not only in the food industry but also in prevention programs for public health

Anisakis and Hysterothylacium species in Mediterranean and North-East Atlantic fishes commonly consumed in Spain: Epidemiological, molecular and morphometric discriminant analysis

The consumption of raw fish parasitized with larval ascaridoid nematodes of the family Anisakidae can cause anisakiasis, provoking gastrointestinal and/or allergic symptomatology. The main causative agents in the Anisakis genus are the sibling species Anisakis simplex sensu stricto (s.s.) and A. pegreffii of the A. simplex sensu lato (s.l.) complex. Larvae of A. simplex (s.l.) are frequently detected in fish commonly consumed in Spain, as are larvae of the genus Hysterothylacium of the family Raphidascarididae, associated with allergic reactions but not considered pathogenic. Reported here are the results of an epidemiological survey of ascaridoid larvae in three commonly consumed fish species in Spain, horse mackerel (Trachurus trachurus) (n = 52), blue whiting (Micromesistius poutassou) (n=93) and anchovy (Engraulis encrasicolus) (n=69), caught in the North-Eastern Atlantic, West Mediterranean and Adriatic Sea. The larvae found in the dissected fish were identified in the following order of abundance: A. simplex (s.l.) (n=2003), Hysterothylacium aduncum (n=422), H. fabri (n=180) and A. physeteris (n=15). Binomial regression analysis showed a correlation between A. simplex (s.l.) and Hysterothylacium larvae abundance and the host geographical location, the North-Eastern Atlantic being the area with the highest parasitation. Fish length and weight and Fulton's condition factor were correlated with A. simplex (s.l.) abundance only in horse mackerel. There was a significant presence of A. simplex (s.l.) and H. aduncum larvae in the musculature of North-Eastern Atlantic blue whiting, the most parasitized part being the anteroventral region, followed equally by the anterodorsal and central sections. The ITS rDNA of larvae of the sibling species A. simplex (s.s.) and A. pegreffii was identified by PCR-RFLP, and a binary logistic regression model was developed to study their morphometric differentiation. Anisakis simplex (s.s.) was detected in the North- Eastern Atlantic and A. pegreffii in all the areas studied. The morphometric analysis discriminated between the two species at the third and fourth larval stages (L3 and L4), the latter obtained by in vitro culture in RPMI-1640 medium. Two discriminant functions were obtained for the L3 and L4 larvae, the ventricle being a key parameter for specific differentiation in both stages, providing taxonomical criteria that could be used besides molecular identification. The present study reveals differences in the parasitation of the studied fish, including the distribution of larvae in the musculature, related to the host species and its geographical origin

Hybrid Genotype of Anisakis simplex (s.s.) and A. pegreffii Identified in Third- and Fourth-Stage Larvae from Sympatric and Allopatric Spanish Marine Waters

The sibling species Anisakis simplex (s.s.) and Anisakis pegreffii are parasites of marine mammals and fish worldwide and the main causative agents of human anisakiasis. In sympatric areas, a hybrid genotype between the two species has been identified, mainly in third-stage larvae, but rarely in fourth-stage and adult forms. The aim of this study was to confirm the presence of hybrid genotypes in larvae parasitizing fish caught in sympatric and allopatric Spanish marine waters, the North-East Atlantic and West Mediterranean, respectively, and to study possible differences in the growth behaviour between genotypes. Of the 254 molecularly analysed larvae, 18 were identified as hybrids by PCR-RFLP analysis of the rDNA ITS region, 11 of which were subsequently confirmed by EF1 α-1 nDNA gene sequencing. These results therefore indicate an overestimation of hybrid genotypes when identification is based only on the ITS region. We also report the detection of a hybrid specimen in a host from the West Mediterranean, considered an allopatric zone. Additionally, fourth-stage larvae with a hybrid genotype were obtained in vitro for the first time, and no differences were observed in their growth behaviour compared to larvae with A. simplex (s.s.) and A. pegreffii genotypes

First Molecular Diagnosis of Clinical Cases of Gastric Anisakiosis in Spain

Anisakiosis is a fish-borne disease with gastrointestinal and/or allergic symptoms caused by the consumption of raw or undercooked fish parasitized with nematode larvae of the genus Anisakis. In Europe, Anisakis pegre i has been detected as the causative agent, although the sibling species Anisakis simplex sensu stricto (s.s.) is also known to cause the disease in other parts of the world, and discrepancies exist regarding their respective pathogenic potential. In Spain a high number of cases has been recorded, with marinated anchovies being the main source of infection, although no specific diagnosis has been documented in humans. In this study, we analyzed three cases of anisakiosis in patients from Barcelona (Spain) who had consumed undercooked hake. All patients described epigastric pain and several larval nematodes were removed endoscopically from their stomachs. Larvae were morphologically characterized as third-stage larvae of Anisakis simplex sensu lato (s.l.) and molecularly identified as A. simplex (s.s.) by means of PCR RFLP of the ITS region of the rDNA and sequencing of the elongation factor1 alpha1 (EF1 -1) nDNA gen. This study represents the first specific identification of Anisakis larvae in clinical cases of anisakiosis reported in Spain. Specific molecular diagnosis is of crucial importance for assessing the health risk of Anisakis sibling species. Hake consu