Caracterització morfològica i molecular i estudi epidemiològic d'espècies d'anisakis i hysterohylacium (nematoda, ascaridoidea)

Resum

ABSTRACT This study is focused on ascaridoid nematodes belonging to the Anisakidae and Raphidascarididae families, parasites of marine fish worldwide, which are of sanitary and/or commercial interest. In the family Anisakidae, species of the genus Anisakis are the main causative agents of human anisakiasis, an emerging disease transmitted by the consumption of raw or undercooked fish parasitized with larvae, which can cause gastrointestinal and/or allergic symptoms. Also, the genus Hysterothylacium of the family Raphidascarididae includes species that, although considered non-pathogenic, have been associated with allergic reactions. In their larval stage, species of both genera can infect a wide range of fish, which act as paratenic or transport hosts, with high prevalence and abundance values. The specific identification is difficult, given the existence of morphologically undistinguishable sibling species in Anisakis and identical morphotypes with different genotypes in Hysterothylacium. The main objective of the present work was to morphologically and molecularly characterize species of these genera detected in three commonly consumed fish in Spain, horse mackerel (Trachurus trachurus), blue whiting (Micromesistius poutassou) and anchovy (Engraulis encrasicolus), as well as to study the main epidemiological factors influencing the degree of parasitation. Another aim was to specifically identify the Anisakis larvae responsible for two clinical cases of anisakiosis, as to date no specific diagnosis of the disease has been documented in Spain. For the study, fish were acquired at Barcelona markets of the three aforementioned species, caught in the North-East Atlantic, the West Mediterranean and the Adriatic Sea. The 2621 larvae detected corresponded mostly to third stage larvae (L3) of A. simplex sensu lato and to a lesser extent to L3 of H. aduncum, both species being observed in all hosts and areas studied. Fourth stage larvae (L4) of H. fabri were also identified in West Mediterranean fish, mainly in horse mackerel, as well as a few L3 larvae of A. physeteris principally in Mediterranean hosts. The epidemiological analysis identified geographical origin as the most influential factor in the abundance of ascaridoid nematodes in the studied fish. Prevalence and abundance were highest in North-Eastern Atlantic hosts: between 79,5 and 100% for A. simplex (s.l.) and 63 and 68% for H. aduncum, with an abundance of about 30 and 5 larvae, respectively. Both species were also widely detected in the musculature of Atlantic blue whiting, with the same distribution pattern, the anteroventral part or belly flaps being the most parasitized portion. A. simplex (s.l.) is a complex of sibling species that includes A. simplex (s.s.) and A. pegrefii, which are morphologically indistinguishable in their larval forms. Molecular analysis by PCR-RFLP of the rDNA ITS region identified A. simplex (s.s.) larvae in North-East Atlantic hosts and A. pegreffii in all hosts and areas studied. Some specimens with a hybrid genotype between the two sibling species were also identified. The number of detected hybrids was reduced by about a third after sequencing of the EF1 α-1 gene of the nDNA, verifying an overestimation of hybrids based on analysis of the ITS region. For the first time, a hybrid specimen was identified in the West Mediterranean, which is considered an allopatric area, the rest being detected in the sympatric area of the North East Atlantic. Larvae of A. simplex (s.s.) and A. pegreffii were also differentiated by a morphometric analysis. L3 larvae from fish and L4 larvae from optimized in vitro culture were studied. Using binary logistic regression, two discriminant functions were obtained, for L3 and L4, which included three and four morphological parameters, respectively. Measurements of the ventricle were considered as key variables, as they were included in both functions. The morphometric analysis discriminated between L3 and L4 of the two species in 78 and 83% of cases, respectively, without significant differences from the molecular identification. Regarding Hysterothylacium, as some species have larvae with the same morphology, it is important to characterize the genotype as well as describe the morphotype. L3 larvae of H. aduncum isolated from fish and L4 larvae and adults obtained from an in vitro optimized culture were subjected to morphotype and genotype analysis. L3 and L4 larvae of H. aduncum isolated in all studied areas corresponded to morphotypes VIII and IX, respectively, of the 18 morphotypes of Hysterothylacium described worldwide. Sequencing of the ITS region permitted the identification of a genotype for the first time in this species, found in both morphotypes. In H. fabri, L3 and L4 larvae corresponded to morphotypes III and IV, respectively, and shared the same genotype, also described here for the first time. Finally, the causative agent of two anisakiasis cases in Barcelona was specifically identified as A. simplex (s.s.) by PCR-RFLP and sequencing. This is the first specific identification of Anisakis larvae in a Spanish clinical case. Specific diagnosis in human anisakiasis is important to understand if differences in pathology are species-related.