1. Investigación

El pescado tiene una relevancia fundamental en la dieta de los seres humanos, sin embargo, su consumo no está exento de riesgos, entre ellos, la presencia de parásitos responsables de enfermedades zoonósicas. Los peces marinos pueden albergar parásitos unicelulares emergentes, de origen terrestre y transmisión hídrica (EWUP, siglas en inglés), sin estar necesariamente infectados. Por otro lado, los peces marinos son verdaderos hospedadores de parásitos de reconocida importancia en salud pública, destacando el nematodo Anisakis pegreffii en el Mediterráneo, causante de la anisakiosis. La primera parte de esta tesis doctoral se enfoca en la detección y caracterización molecular (PCR y metabarcoding) de algunos EWUP de importancia en peces de consumo (cultivados y silvestres), de la costa española del Mediterráneo. Se detectaron especies y subtipos zoonósicos de Cryptosporidium, Blastocystis y microsporidios, aunque en prevalencias muy bajas, sugiriendo un limitado riesgo de transmisión por consumo de pescado procedente de las poblaciones estudiadas. En la segunda parte, se propone un protocolo in vitro para el desarrollo larvario temprano de A. pegreffii, complementado con un estudio ultraestructural. Esta herramienta, como parte del ciclo biológico in vitro de A. pegreffii, puede ser muy útil para la obtención de material biológico destinado a futuros estudios de patogenicidad, diagnóstico o terapéutica.

The in vitro life cycle of zoonotic helminths is an essential tool for -omic translational studies focused on disease control and treatment. Anisakiosis is an emerging zoonosis contracted by the ingestion of raw or undercooked fish infected with the third stage larvae (L3) of two sibling species Anisakis simplex sensu stricto (s.s.) and Anisakis pegreffii, the latter being the predominant species in the Mediterranean basin. Recently, in vitro culture of A. pegreffii has been developed to enable fast and large-scale production of fertile adults. However, the conditions for larval development from hatching to infective L3 were not fulfilled to complete the cycle. Herein, we used a Drosophila medium supplemented with chicken serum and adjusted different osmolarities to maintain the culture of L3 hatched from eggs for up to 17 weeks. The highest survival rate was observed in the medium with the highest osmolarities, which also allowed the highest larval exsheathment rate. Key morphological features of embryogenesis and postembryogenesis studied by transmission electron microscopy revealed that the excretory gland cell is differentiated already up to 48 h post-hatching. Extracellular vesicles and cell-free mitochondria are discharged between the two cuticle sheets of the second stage larvae (L2). Contemporarly cultivated, two populations of adult A. simplex s.s. and A. pegreffii reached an average production of 29,914.05 (± 27,629.36) and 24,370.96 (± 12,564.86) eggs/day/female, respectively. The chromosome spreads of A. pegreffii obtained from mature gonads suggests a diploid karyotype formula of 2n = 18. The development of a reliable protocol for the in vitro culture of a polyxenous nematode such as Anisakis spp. will serve to screen for much needed novel drug targets, but also to study the intricated and unknown ecological and physiological traits of these trophically transmitted marine nematodes.

Microsporidia are widely spread obligate intracellular fungal pathogens from vertebrate and invertebrate organisms, mainly transmitted by contaminated food and water. This study aims to detect the presence of major human-pathogenic microsporidia, i.e., Enterocytozoon bieneusi, Encephalitozoon intestinalis, Encephalitozoon hellem, and Encephalitozoon cuniculi, in the gastrointestinal tract of commercially harvested marine fish from Mediterranean coast of the Comunidad Valenciana, Eastern Spain. A total of 251 fish, 138 farmed fish and 113 wild fish from commercial fishing were tested by SYBR Green real-time PCR, enabling the simultaneous detection of the four targeted species. E. intestinalis/hellem was found in 1.45% of farmed fish and 7.96% of wild fish, while Enterocytozoonidae was detected in 2.90% and 18.58% of farmed and wild fish, respectively. E. cuniculi was not detected in any of the analyzed specimens. To the authors’ knowledge, this is the first report of E. intestinalis/hellem in fish, particularly in marine fish. Although the role of fish in these species’ epidemiology remains unknown, this finding points out a potential public health risk linked to fish consumption. Further studies are necessary to characterize these microsporidia in fish hosts better and to elucidate their epidemiological role.