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Phlebotomus perniciosus is a major vector of Leishmania infantum in the Mediterranean. While the seroprevalence of leishmaniosis in Spanish dogs and cats has been studied, data on the exposure of cats to P. perniciosus bites under natural conditions without repellents is limited. Stray cats could serve as sentinels for L. infantum and P. perniciosus exposure. This study analyzed sera from 204 apparently healthy stray cats, collected from January 2021 to January 2022, for antibodies against P. perniciosus saliva and L. infantum parasites. Anti-sand fly antibodies were detected in 40.69% of cats using an ELISA with the recombinant salivary protein SP03B of P. perniciosus . Seroprevalence of L. infantum infection was 23.52% by Western blot and 27.41% by ELISA, with an overall seroprevalence of 40.69% (95% CI 34.18-47.54%). This is the first assessment of antibody response to P. perniciosus saliva and L. infantum in naturally exposed stray cats in Spain. Further research is needed to examine the salivary antigens recognized by cats and to explore the relationship between P. perniciosus exposure and L. infantum infection severity in cats.

Leishmania infantum is the primary cause of visceral and cutaneous leishmaniasis in the European Mediterranean region. Subspecies-level characterization of L. infantum aids epidemiological studies by offering insights into the evolution and geographical distribution of the parasite and reservoir identity. In this study, conducted in north-east Spain, 26 DNA samples of L. infantum were analyzed, comprising 21 from 10 humans and 5 from 5 dogs. Minicircle kinetoplast DNA (kDNA) polymerase chain reaction assays using primers MC1 and MC2, followed by sequencing, were employed to assess intraspecific genetic variability. Single-nucleotide polymorphism (SNP) analysis detected seven genotypes (G1, G2, G12*-G15*, and G17*), with five being reported for the first time (*). The most prevalent was the newly described G13 (54%), while the other currently identified genotypes were predominantly found in single samples. The in silico restriction fragment length polymorphism (RFLP) method revealed five genotypes (B, F, N, P, and W), one of them previously unreported (W). Genotype B was the most prevalent (85%), comprising three SNP genotypes (G1, G2, and G13), whereas the other RFLP genotypes were associated with single SNP genotypes. These kDNA genotyping methods revealed significant intraspecific genetic diversity in L. infantum , demonstrating their suitability for fingerprinting and strain monitoring.

The European conger, Conger conger , is a benthic marine fish species with a geographical distribution extending through the northeastern Atlantic and the Mediterranean. Despite being extensively distributed and widely appreciated by Spanish consumers, studies regarding parasite presence in this fish are scarce. In the present work, a hundred and eight specimens from the Mediterranean coast of northeastern Spain (Catalan waters) were surveyed for the presence of nematode parasites. Several species were morphologically identified: third-stage larvae of Anisakis type I (sensu Berland, 1961) ( n = 131), third-stage larvae of Anisakis type II (sensu Berland, 1961) ( n = 18), third- and fourth-stage larvae and adults of Hysterothylacium spp. ( n = 48), adults of Cucullanus sp. ( n = 391), and adults of Cristitectus congeri ( n = 69). Moreover, some fish and decapode species were also observed as part of the host's diet, with the most detected preys being Micromesistius poutassou , Sardina pilchardus , Macropipus sp., and Goneplax rhomboides . This represents the first survey of nematode parasites infecting C. conger from the northeastern Spanish Mediterranean waters. Among the parasite species detected, the presence of Anisakis species should be highlighted as the ingestion of C. conger parasitized with these larvae could potentially lead to anisakiasis in consumers.

Understanding the genomic underpinnings of thermal adaptation is a hot topic in eco-evolutionary studies of parasites. Marine heteroxenous parasites have complex life cycles encompassing a free-living larval stage, an ectothermic intermediate host and a homeothermic definitive host, thus representing compelling systems for the study of thermal adaptation. The Antarctic anisakid Contracaecum osculatum sp. D is a marine parasite able to survive and thrive both at very cold and warm temperatures within the environment and its hosts. Here, a de novo transcriptome of C. osculatum sp. D was generated for the first time, by performing RNA-Seq experiments on a set of individuals exposed to temperatures experienced by the nematode during its life cycle. The analysis generated 425,954,724 reads, which were assembled and then annotated. The high-quality assembly was validated, achieving over 88% mapping against the transcriptome. The transcriptome of this parasite will represent a valuable genomic resource for future studies aimed at disentangling the genomic architecture of thermal tolerance and metabolic pathways related to temperature stress., (© 2023. Springer Nature Limited.)

The second-line antileishmanial compound pentamidine is administered intramuscularly or, preferably, by intravenous infusion, with its use limited by severe adverse effects, including diabetes, severe hypoglycemia, myocarditis and renal toxicity. We sought to test the potential of phospholipid vesicles to improve the patient compliance and efficacy of this drug for the treatment of leishmaniasis by means of aerosol therapy. The targeting to macrophages of pentamidine-loaded liposomes coated with chondroitin sulfate or heparin increased about twofold (up to ca. 90%) relative to noncoated liposomes. The encapsulation of pentamidine in liposomes ameliorated its activity on the amastigote and promastigote forms of Leishmania infantum and Leishmania pifanoi , and it significantly reduced cytotoxicity on human umbilical endothelial cells, for which the concentration inhibiting 50% of cell viability was 144.2 ± 12.7 µM for pentamidine-containing heparin-coated liposomes vs. 59.3 ± 4.9 µM for free pentamidine. The deposition of liposome dispersions after nebulization was evaluated with the Next Generation Impactor, which mimics human airways. Approximately 53% of total initial pentamidine in solution reached the deeper stages of the impactor, with a median aerodynamic diameter of ~2.8 µm, supporting a partial deposition on the lung alveoli. Upon loading pentamidine in phospholipid vesicles, its deposition in the deeper stages significantly increased up to ~68%, and the median aerodynamic diameter decreased to a range between 1.4 and 1.8 µm, suggesting a better aptitude to reach the deeper lung airways in higher amounts. In all, nebulization of liposome-encapsulated pentamidine improved the bioavailability of this neglected drug by a patient-friendly delivery route amenable to self-administration, paving the way for the treatment of leishmaniasis and other infections where pentamidine is active.

Sibling species of the Contracaecum rudolphii (s.l.) complex are habitual endoparasites of cormorants of the Phalacrocoracidae family, worldwide. In Europe, the two species, C. rudolphii sp. A and C. rudolphii sp. B, have been identified. However, information regarding the occurrence and distribution of these anisakids in cormorants from Spain is scarce. In the present study, 20 specimens of the European Shag, Ph. aristotelis desmarestii , from the western Mediterranean Spanish marine coast were parasitologically analyzed for the presence of nematodes. All hosts were found parasitized with Contracaecum specimens ( n = 1,517). A representative subsample was genetically identified as C. rudolphii sp. A by sequence analysis of the mtDNA cox 2 gene and the ITS1 and ITS2 regions of the rDNA. This represents the first report of C. rudolphii sp. A from the Spanish Mediterranean waters. Population genetic analysis was performed including other C. rudolphii sp. A specimens from the west Sardinian and the Tyrrhenian Sea. At the intraspecific level, a significant genetic differentiation ( Fst ≈ 0.08, p < 0.00001) between the metapopulation from the Spanish Mediterranean coast and that from the Sardinian waters was observed; whereas, no differentiation was found between metapopulations of the parasite from the Spanish and the Tyrrhenian Italian coast. The findings highly support the hypothesis of the adaptation of the life cycle of C. rudolphii sp. A in brackish and marine ecosystems. Furthermore, the results on the population genetics of C. rudolphii sp. A suggest the possible role of the migration routes of wintering populations of cormorants in the Mediterranean Sea in influencing the parasite genetic structure., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Roca-Geronès, Fisa, Montoliu, Casadevall, Tobella, Bas, Palomba and Mattiucci.)

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.

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 (R 2 = 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 (R 2 = 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. © 2020 Society of Chemical Industry., (© 2020 Society of Chemical Industry.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interests., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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 pegreffii 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 α-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 consumption stands out as a risk factor for anisakiosis, since this fish species can be highly parasitized.

The presence of zoonotic Hysterothylacium larvae in fish from Spanish Atlantic and Mediterranean waters, which can cause economic losses for commercial fisheries, has been reported in several studies; however, little is known about species identity in this region. The aim of this study was to identify at species level the Hysterothylacium morphotypes detected in three commonly consumed fish: horse mackerel (Trachurus trachurus), blue whiting (Micromesistius poutassou) and anchovy (Engraulis encrasicolus). Third- and fourth-stage Hysterothylacium larvae, as well as adults obtained from larval in vitro culture, were morphologically and molecularly identified by ITS1/ITS2 rDNA sequencing. Four Hysterothylacium morphotypes were detected. Genetic analysis showed that morphotypes VIII and IX were different larval stages of Hysterothylacium aduncum, which was supported by cultured adult species identification. Morphotypes III and IV were found to correspond to different developmental stages of another species of Hysterothylacium. As all larval types detected were morphologically indistinguishable from others previously reported yet showed clear genetic differences, they are referred here as new genotypes. This is the first time that ITS-sequence data of various developmental stages of the same species, including adults, have been studied and compared, providing crucial knowledge for future studies on Hysterothylacium identification and biology., (© 2018 John Wiley & Sons Ltd.)

The extensive presence of anisakids in fish for human consumption has become a problem of food safety and quality. The aim of this study was to develop and assess the performance of a quantitative SYBR Green qPCR assay for the detection and quantification of Anisakis DNA in fish by-products. L3 nematode larvae of A. simplex (s.l.) (n=510), A. physeteris (n=3), Hysterothylacium sp. (n=10) and Pseudoterranova sp. (n=1), isolated from blue whiting, horse mackerel and monkfish, were used for the optimization of the molecular assay. In addition, molecularly typed larvae of A. simplex (s.s.) (n=10) and A. pegreffii (n=5) of the complex A. simplex (s.l.) were used for the specificity assay. Primers targeting the mitochondrial cytochrome c oxidase subunit II gene (COII) were selected. Analytical sensitivity and reproducibility were evaluated in a food matrix consisting of commercial fish-derived food spiked with larvae of A. simplex (s.l.). The assay proved to be specific for the three analyzed Anisakis species. A high reproducibility and sensitivity was detected, with a 95% limit of detection (LOD) of 0.30ng ( 95% CI 0.15-1.50) of A. simplex (s.l.) DNA per gram of food matrix and an operative LOD of 1.50ng after a PROBIT analysis. The assay was applied to study the presence of Anisakis in four types of processed commercial food, namely crab sticks, "gulas", croquettes and burgers. Overall, 180 food samples from 15 commercial brands were studied, detecting Anisakis DNA in over half of them. The analyzed surimi-based products, "gulas" and crab sticks, showed the highest Anisakis burden (5.86±0.69 and 4.68±0.73ng of Anisakis DNA per gram of food, respectively). Our results indicate that the optimized SYBR Green qPCR technique is an accurate and sensitive method that may improve detection of Anisakis in fresh and processed products., (Copyright © 2017 Elsevier B.V. All rights reserved.)