A case report of Anisakis pegreffii (Nematoda, Anisakidae) identified from archival paraffin sections of a Croatian patient.

BACKGROUND: This is the first report of anisakiasis in a Croatian patient, evidenced from an archival paraffin-embedded and hematoxylin-eosin stained tissue section. Anisakiasis has been only suspected in the country based on previously detected anti-Anisakis IgE seroprevalence in the healthy coastal population, as well as an acute case where pathohistological and serological findings suggested the diseases, but the migrating larva has not been retrieved. CASE PRESENTATION: Seventy years-old female, operated in 1998 for pulmonary carcinoma, was admitted to the General hospital Sibenik, Croatia in 2003, because of gastric pain and nausea that lasted for couple of days. She was showing good general condition, full mobility and lucidity, subfebrile status. Abdominal palpation inferred acute pain in paraumbilical and ileocecal region. Exploratory right pararectal laparotomy revealed a hardened, 5 cm-long structure, located intraluminally in the sigmoid colon, not perforating colon serosa. The process has been dissected and sent for patohistological diagnosis. Results showed a 2 mm-long whitish nematode spiralised in muscular layer of colon mucosa surrounded by granulomatous inflammation. CONCLUSION: After genomic DNA isolation of the nematode from the histological section, and amplification at the mitochondrial cytochrome oxidase 2 locus, etiological agent has been identified as Anisakis pegreffii. Used methodology suggests that screening of archival suspicious sections is feasible in order to study epidemiology of this zoonotic disease poorly recognised in Croatia.

Role of biogenic amines in the post-mortem migration of Anisakis pegreffii (Nematoda: Anisakidae Dujardin, 1845) larvae into fish fillets.

Infective third-stage larvae (L3) of nematode Anisakis spp. have been recognized as one of the major food-borne threats in lightly processed fish products in Europe, particularly in the Mediterranean region. Therefore, the effect of different storage temperatures of fish on larval post-mortem migration from visceral cavity into fillets is an important parameter to take into account when evaluating the risk for consumer safety. The European anchovy (Engraulis encrasicolus) were caught during fishing season, a subsample of fillets was checked for the presence of Anisakis larvae at capture (mean abundance=0.07), and the rest was stored at four different temperatures (-18, 0, 4 and 22°C) in order to count migrating larvae and measure the production of biogenic amines over a period of time. Larvae were identified by morphological features and molecular tools. Post-mortem migration was observed in fillets stored at 0 and 4°C after three and five days, respectively, but not at 22 and -18°C. In case of storage at 22°C for two days, at the onset of putrefaction of the visceral organs, larvae migrated out of the visceral cavity towards the fish surface. Measured pH and biogenic amine profile during storage indicated that certain biochemical conditions trigger larval migration into fillets. Likewise, migration was observed at pH ~6.4 when sensory degradation of the fish was markedly visible. Although larval migration was delayed for approximately four days at a temperature of <4°C the correlation between pH and abundance of A. pegreffii larvae in the fillet was high and statistically significant at both 0 (r=0.998, p<0.01) and 4°C (r=0.946, p<0.05). Out of eight biogenic amines measured, cadaverine and putrescine levels correlated the most with the post-mortem migration at 4°C, while tyramine levels were significant at both temperatures.

Anti-Anisakis IgE seroprevalence in the healthy Croatian coastal population and associated risk factors.

BACKGROUND: The main objective of the study was to determine the degree of sensitization to Anisakis spp. antigens in healthy coastal population of Dalmatia given the high thermally unprocessed fish intake rate present in this area, suggested as a significant risk factor for anisakiasis. We performed a monocenter, cross-sectional pilot study stratified by geographic area of residence, conducted at the County secondary healthcare provider Medicine-biochemical Laboratory in Split (Croatia), from November 2010 till December 2011, on 500 unpaid volunteer subjects undergoing routine blood analysis and belonging to the south coast of the Adriatic Sea. METHODOLOGY/PRINCIPAL FINDINGS: We studied the IgE seroprevalence to Anisakis spp. Ani s l and Ani s 7 allergens by indirect ELISA in healthy subjects, which were selected at random in the region of Dalmatia (Southern Croatia), among islands, coastal urban and inland rural populations. In order to detect possible cross-reactivity to other human helminthes, serum samples were tested also for the presence of IgG antibodies to Ascaris lumbricoides and Toxocara canis. The overall and coastal Anisakis seroprevalences for the sampled population were 2% and 2.5%, respectively. The logistic univariate regression analysis confirmed that regarding anti-Anisakis IgE seroprevalence, raw fish intake, daily fish intake, homemade origin of fish dish and occupational contact (professional, artisanal or hobby contact with fishery or fish industry) were risk factors associated to Anisakis spp. sensitization, but neither of the variables was exclusive for a particular seropositive population. Also, a significant difference was observed between seropositive and seronegative subjects that had stated allergy or symptoms associated with allergy (atopic dermatitis, asthma or rhinitis) in their previous history. CONCLUSIONS/SIGNIFICANCE: Being the first in Croatia, our study underlines the necessity of incorporating Anisakis spp. allergens in routine hypersensitivity testing of coastal population.

As, Cd, Hg and Pb in four edible shellfish species from breeding and harvesting areas along the eastern Adriatic Coast, Croatia.

Four species of shellfish (Mytilus galloprovincialis, Ostrea edulis, Chlamis varia and Venus verrucosa) were collected during the autumn 2011 and spring 2012 along the eastern Adriatic coast from six shellfish harvesting areas (all species) and 13 breeding sampling areas (mussels) to assess As, Cd, Hg and Pb levels and the human risks of shellfish consumption. The mean metal concentrations (wet weight) in the examined species ranged from 1.420 to 9.575mg/kg for As, 0.034 to 1.270 for Cd, 0.005 to 0.680 for Hg and 0.140 to 2.072 for Pb. Examination of the spatial distribution of As, Cd, Hg and Pb revealed statistically significant differences among the studied areas. Since the concentrations were below the maximum prescribed by the laws of the EU and Croatia (the concentrations slightly exceeded the upper limits for three samples; Pb, Cd and Hg) and the hazard index, (HI) for Cd, Hg and Pb were below 1 and the target cancer risk (TR) for As was lower than 1×10(-6), there is no human health risk of consumption of shellfish from Croatian waters.

Ecology and genetic structure of zoonotic Anisakis spp. from adriatic commercial fish species.

Consumption of raw or thermally inadequately treated fishery products represents a public health risk, with the possibility of propagation of live Anisakis larvae, the causative agent of the zoonotic disease anisakidosis, or anisakiasis. We investigated the population dynamics of Anisakis spp. in commercially important fish-anchovies (Anisakis), sardines (Sardina pilchardus), European hake (Merluccius merluccius), whiting (Merlangius merlangus), chub mackerel (Scomber japonicus), and Atlantic bluefin tuna (Thunnus thynnus)-captured in the main Adriatic Sea fishing ground. We observed a significant difference in the numbers of parasite larvae (1 to 32) in individual hosts and between species, with most fish showing high or very high Anisakis population indices. Phylogenetic analysis confirmed that commercial fish in the Adriatic Sea are parasitized by Anisakis pegreffii (95.95%) and Anisakis simplex sensu stricto (4.05%). The genetic structure of A. pegreffii in demersal, pelagic, and top predator hosts was unstructured, and the highest frequency of haplotype sharing (n = 10) was between demersal and pelagic fish.

Molecular identification and population dynamic of Anisakis pegreffii (Nematoda: Anisakidae Dujardin, 1845) isolated from the European anchovy (Engraulis encrasicolus L.) in the Adriatic Sea.

Anchovy Engraulis encrasicolus (L.) is a coastal pelagic and euryhaline species that represents the only European species of the family Engraulidae, with a widespread distribution. In Croatia, it is marketed fresh, frozen, salted or marinated and mainly exported to Italy and Spain, however Anisakis sp. larval infection is frequently the reason for border rejection. Since it is known that the prevalence and intensity of Anisakis infection varies with fish species, fishing area and season, the aim of our study was to identify Anisakis sp. parasitizing European anchovy and infer its population dynamic through a 2.5-year period. Larvae were found coiled and encysted on the external wall of intestine (94%) and reproductive organs (6%), rarely in fillets. Prevalence was 76.1% (95% confidence limits 74.51-77.56%), mean abundance 6.59 (bootstrap 95% confidence limits 5.81-7.26) and mean intensity 8.67 (bootstrap 95% confidence limits 7.82-9.35). The partial CO2 mitochondrial DNA sequence of the isolated anisakids confirmed clustering of the anchovy parasite within A. pegreffii sister group. Parasite population structure showed plasticity inferred by fishing ground, sampling year and fish gender and size. Compared to anisakid prevalence/abundance in other fish, the European anchovy in the Adriatic Sea represents a moderately high-infected paratenic host, although in the Mediterranean and Atlantic waters, anchovies have shown strikingly lesser values of prevalence. Since this host represents one of the most attractive Mediterranean fisheries products traditionally consumed without thermal preparation that in any case would not disrupt larval antigenicity and prevent human allergies, and given the high prevalence of the anisakid within the host, it is necessary to include anchovy into more firm risk assessment frames in order to develop measures that will support the safe alimentary production and consumption of seafood.