Fecundity, in vitro early larval development and karyotype of the zoonotic nematode Anisakis pegreffii.

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 in Commercially Harvested Marine Fish: A Potential Health Risk for Consumers.

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.

Molecular Detection and Epidemiology of Potentially Zoonotic Cryptosporidium spp. and Giardia duodenalis in Wild Boar (Sus scrofa) from Eastern Spain.

The protozoans Giardia duodenalis and Cryptosporidium spp. are common causes of gastrointestinal disease in humans and animals. While both are commonly documented in domestic animals, few studies have analysed their presence in wildlife. To assess the prevalence of both parasites in wild boar (Sus scrofa) in the Valencian Community (eastern Spain), 498 wild boar faecal samples were collected from 2018 to 2022. Cryptosporidium spp. was detected by performing a nested PCR targeting a 578 bp sequence of the small subunit ribosomal RNA gene (SSU rRNA), followed by sequencing and phylogenetic analysis. For G. duodenalis, a qPCR amplifying a fragment of 62 bp from the SSU rRNA was employed. Positive samples were genotyped for glutamate dehydrogenase and ß-giardin genes. Different epidemiological factors were considered potential modulating variables in the transmission of both parasites. G. duodenalis prevalence was 1.20%, while Cryptosporidium spp. prevalence reached 21.7%. Coinfection was observed in 0.2%. Genotyping of G. duodenalis isolates only detected genotype E. Two species of Cryptosporidium spp. were identified: Cryptosporidium scrofarum and Cryptosporidium suis. The results of this study demonstrate that the exposure to Cryptosporidium spp. in wild boars is high, particularly among young individuals belonging to the Typical Mediterranean climate. Moreover, the probability of infection is dependent on both the season and the density of wild boars. On the other side, exposure to G. duodenalis seems scarce and is influenced, in turn, by the climate. Both Cryptosporidium species detected in the present study have been reported in humans. Due to wild boar increasing in number and their colonisation of urban and peri-urban areas, this could represent an inherent health risk for the human population.

Molecular Characterization of Cryptosporidium spp. in Cultivated and Wild Marine Fishes from Western Mediterranean with the First Detection of Zoonotic Cryptosporidium ubiquitum.

Fish not only harbor host-specific species/genotypes of Cryptosporidium, but also species like zoonotic C. parvum or anthroponotic C. hominis, which can pose a risk for fish consumers. This study aims to investigate fish cryptosporidiosis in an important aquaculture and fishery area of the Western Mediterranean (Comunidad Valenciana, Spain). We analyzed 404 specimens belonging to the following three groups: cultivated fish (N = 147), wild synanthropic fish (N = 147) and wild fish from extractive fisheries (N = 110). Nested PCR targeting the 18S rRNA gene, followed by sequencing and phylogenetic analysis, were performed. Positive isolates were also amplified at the actin gene locus. An overall prevalence of 4.2% was detected, with the highest prevalence in the synanthropic group (6.1%). C. molnari was identified in thirteen specimens from seven different host species. Zoonotic C. ubiquitum was detected in two European sea bass (Dicentrarchus labrax). One isolate similar to C. scophthalmi was detected in a cultivated meagre (Argyrosomus regius), and one isolate, highly divergent from all the Cryptosporidium species/genotypes described, was identified from a synanthropic round sardinella (Sardinella aurita). This study contributes to increasing the molecular data on fish cryptosporidiosis, expanding the range of known hosts for C. molnari and identifying, for the first time, zoonotic C. ubiquitum in edible marine fishes, pointing out a potential health risk.

Detection of Stephanurus dentatus in wild boar urine using different parasitological techniques.

Stephanurus dentatus is a nematode that parasitizes the urinary tract of domestic and wild Suidae, especially in tropical areas. However, there is a lack of information about stephanurosis in wild boar (Sus scrofa), thus making it necessary to develop sensitive techniques with which to diagnose this pathogen in order to carry out further research. In Spain, the high prevalence of this nematode has been evidenced in Doñana National Park (DNP). The objective of the present work is twofold. The first is to compare the efficacy of three parasitological techniques to detect S. dentatus eggs in the urine of infected wild boar: (i) gravity sedimentation, (ii) sedimentation by centrifugation, and (iii) flotation techniques, while the second is to determine whether the quantification of eggs can serve as an indicative value of the host's parasite intensity. In order to accomplish these purposes, 27 wild boars from DNP were necropsied, and the urinary system of each animal was examined in order to determine parasite intensity. While all the aforementioned techniques can be used to detect eggs in urine, the most effective in terms of egg quantification are sedimentation by gravity and by centrifugation, as they allow a greater number of S. dentatus eggs to be detected. However, none of the results obtained with these techniques significantly correlated with the number of adult nematodes parasitizing the host, signifying that counts in urine can provide guidance on only the parasite intensity of wild boar.

Potential Risk of Three Zoonotic Protozoa (Cryptosporidium spp., Giardia duodenalis, and Toxoplasma gondii) Transmission from Fish Consumption.

In recent decades, worldwide fish consumption has increased notably worldwide. Despite the health benefits of fish consumption, it also can suppose a risk because of fishborne diseases, including parasitic infections. Global changes are leading to the emergence of parasites in new locations and to the appearance of new sources of transmission. That is the case of the zoonotic protozoa Cryptosporidium spp., Giardia duodenalis, and Toxoplasma gondii; all of them reach aquatic environments and have been found in shellfish. Similarly, these protozoa can be present in other aquatic animals, such as fish. The present review gives an overview on these three zoonotic protozoa in order to understand their potential presence in fish and to comprehensively revise all the evidences of fish as a new potential source of Cryptosporidium spp., Giardia duodenalis, and Toxoplasma gondii transmission. All of them have been found in both marine and freshwater fishes. Until now, it has not been possible to demonstrate that fish are natural hosts for these protozoa; otherwise, they would merely act as mechanical transporters. Nevertheless, even if fish only accumulate and transport these protozoa, they could be a "new" source of infection for people.