Fernando Simón’s research while affiliated with University of Salamanca and other places

Background The trematode Fasciola hepatica is the most widespread causative agent of fasciolosis, a parasitic disease that mainly affects humans and ruminants worldwide. During F. hepatica infection, newly excysted juveniles (FhNEJ) emerge in the duodenum of the mammalian host and migrate towards their definitive location, the intra-hepatic biliary ducts. Understanding how F. hepatica traverses the intestinal wall and migrates towards the liver is pivotal for the development of more successful strategies against fasciolosis. The central enzyme of the mammalian fibrinolytic system is plasmin, a serine protease whose functions are exploited by a number of parasite species owing to its broad spectrum of substrates, including components of tissue extracellular matrices. The aim of the present work is to understand whether FhNEJ co-opt the functions of their host fibrinolytic system as a mechanism to facilitate trans-intestinal migration. Methodology/Principal Findings A tegument-enriched antigenic extract of FhNEJ (FhNEJ-Teg) was obtained in vitro, and its capability to bind the zymogen plasminogen (PLG) and enhance its conversion to the active protease, plasmin, were analyzed by a combination of enzyme-linked immunosorbent, chromogenic and immunofluorescence assays. Additionally, PLG-binding proteins in FhNEJ-Teg were identified by bidimensional electrophoresis coupled to mass spectrometry analysis, and the interactions were validated using FhNEJ recombinant proteins. Conclusions/Significance Our results show that FhNEJ-Teg contains proteins that bind PLG and stimulate its activation to plasmin, which could facilitate the traversal of the intestinal wall by FhNEJ and contribute to the successful establishment of the parasite within its mammalian host. Altogether, our findings contribute to a better understanding of host-parasite relationships during early fasciolosis and may be exploited from a pharmacological and/or immunological perspective for the development of treatment and control strategies against this global disease.

Background: The trematode Fasciola hepatica is the most widespread causative agent of fasciolosis, a parasitic disease that mainly affects humans and ruminants worldwide. During F. hepatica infection, newly excysted juveniles (FhNEJ) emerge in the duodenum of the mammalian host and migrate towards the definitive location of the parasite, the intra-hepatic biliary ducts. Understanding how F. hepatica traverses the intestinal wall and migrates towards the liver is pivotal for the development of more successful strategies against fasciolosis. The central enzyme of the mammalian fibrinolytic system is plasmin, a serine protease whose functions are exploited by a number of parasite species owing to its broad spectrum of substrates, including components of tissue extracellular matrices. The aim of the present work is to understand whether FhNEJ co-opt the functions of their host fibrinolytic system as a mechanism to facilitate trans-intestinal migration. Methodology/Principal Findings: An FhNEJ tegument protein extract (FhNEJ-Teg) was obtained in vitro, and its capability to bind the zymogen plasminogen (PLG) and enhance its conversion to the active protease, plasmin, were analyzed by a combination of enzyme-linked immunosorbent, chromogenic and immunofluorescence assays. Additionally, PLG-binding proteins in FhNEJ-Teg were identified by 2D electrophoresis coupled to mass-spectrometry analysis, and the interactions were validated using FhNEJ recombinant proteins. Conclusions/Significance: Our results show that FhNEJ-Teg contains proteins that bind PLG and stimulate its activation to plasmin, which could facilitate the traversal of the intestinal wall by FhNEJ and contribute to the successful establishment of the parasite within its mammalian host. Altogether, our findings contribute to a better understanding of host-parasite relationships during early fasciolosis and may be exploited from a pharmacological and/or immunological perspective for the development of treatment and control strategies against this global disease.

Fasciolosis caused by the trematode Fasciola hepatica is a zoonotic neglected disease affecting animals and humans worldwide. Infection occurs upon ingestion of aquatic plants or water contaminated with metacercariae. These release the newly excysted juveniles (FhNEJ) in the host duodenum, where they establish contact with the epithelium and cross the intestinal barrier to reach the peritoneum within 2–3 h after infection. Juveniles crawl up the peritoneum towards the liver, and migrate through the hepatic tissue before reaching their definitive location inside the major biliary ducts, where they mature into adult worms. Fasciolosis is treated with triclabendazole, although resistant isolates of the parasite are increasingly being reported. This, together with the limited efficacy of the assayed vaccines against this infection, poses fasciolosis as a veterinary and human health problem of growing concern. In this context, the study of early host-parasite interactions is of paramount importance for the definition of new targets for the treatment and prevention of fasciolosis. Here, we develop a new in vitro model that replicates the first interaction between FhNEJ and mouse primary small intestinal epithelial cells (MPSIEC). FhNEJ and MPSIEC were co-incubated for 3 h and protein extracts (tegument and soma of FhNEJ and membrane and cytosol of MPSIEC) were subjected to quantitative SWATH-MS proteomics and compared to respective controls (MPSIEC and FhNEJ left alone for 3h in culture medium) to evaluate protein expression changes in both the parasite and the host. Results show that the interaction between FhNEJ and MPSIEC triggers a rapid protein expression change of FhNEJ in response to the host epithelial barrier, including cathepsins L3 and L4 and several immunoregulatory proteins. Regarding MPSIEC, stimulation with FhNEJ results in alterations in the protein profile related to immunomodulation and cell-cell interactions, together with a drastic reduction in the expression of proteins linked with ribosome function. The molecules identified in this model of early host-parasite interactions could help define new tools against fasciolosis.

Fasciola hepatica is a trematode parasite that infects animals and humans causing fasciolosis, a worldwide-distributed disease responsible for important economic losses and health problems. This disease is of growing public health concern since parasite isolates resistant to the current treatment (triclabendazole) have increasingly been described. F. hepatica infects its vertebrate host after ingestion of the encysted parasite (metacercariae), which are found in the water or attached to plants. Upon ingestion, newly excysted juveniles of F. hepatica (FhNEJ) emerge in the intestinal lumen and cross the intestinal barrier, reach the peritoneum and migrate to the biliary ducts, where adult worms fully develop. Despite the efforts made to develop new therapeutic and preventive tools, to date, protection against F. hepatica obtained in different animal models is far from optimal. Early events of host-FhNEJ interactions are of paramount importance for the infection progress in fasciolosis, especially those occurring at the host-parasite interface. Nevertheless, studies of FhNEJ responses to the changing host environment encountered during migration across host tissues are still scarce. Here, we set-up an ex vivo model coupled with quantitative SWATH-MS proteomics to study early host-parasite interaction events in fasciolosis. After comparing tegument and somatic fractions from control parasites and FhNEJ that managed to cross a mouse intestinal section ex vivo, a set of parasite proteins whose expression was statistically different were found. These included upregulation of cathepsins L3 and L4, proteolytic inhibitor Fh serpin 2, and a number of molecules linked with nutrient uptake and metabolism, including histone H4, H2A and H2B, low density lipoprotein receptor, tetraspanin, fatty acid binding protein a and glutathione-S-transferase. Downregulated proteins in FhNEJ after gut passage were more numerous than the upregulated ones, and included the heath shock proteins HSP90 and alpha crystallin, amongst others. This study brings new insights into early host-parasite interactions in fasciolosis and sheds light on the proteomic changes in FhNEJ triggered upon excystment and intestinal wall crossing, which could serve to define new targets for the prevention and treatment of this widespread parasitic disease.

Helminth parasitoses are among the most prevalent health issues worldwide. Their control depends largely on unravelling host–parasite interactions, including parasitic exploitation of the host haemostatic system. The present study undertakes a scoping review of the research carried out in this field with the aim of unifying and updating concepts. Multiple keywords combined with Boolean operators were employed to design the literature search strategy. Two online databases were used to identify original peer-reviewed articles written in English and published before 1st January 2020 describing molecular interactions between helminth parasites and the host haemostatic system. Relevant data from the selected sources of evidence were extracted and analysed. Ninety-six publications reporting 259 interactions were selected. Fifty-three proteins belonging to 32 species of helminth parasites were involved in interactions with components of the host haemostatic system. Many of these proteins from both parasite and host were conserved among the different interactions identified. Most of these interactions were related to the inhibition of the coagulation system and the activation of fibrinolysis. This was associated mainly with a potential of parasites to reduce the formation of blood clots in the host and attributed to biological processes, such as parasite nutrition, survival, invasion, evasion and migration or the appearance of pathological mechanisms in the host. A wide range of helminth parasites have developed similar strategies to exploit the haemostatic system of their hosts, which could be regarded as an evolutionary conserved mechanism that could confer benefits to parasites in terms of survival and establishment in their vertebrate hosts.

Human dirofilariosis is a clinical entity caused by infection with nematode species of the genus Dirofilaria. The traditional picture depicts the disease as a sporadic event associated with the presence of a single immature worm causing a nodular lesion. With the aim to reassess this paradigm, establishing a more accurate picture of the disease and homogenize criteria, a scoping review was conducted by searching, screening and analysing published clinical cases of human dirofilariosis worldwide during the 21st century. After extracting data from 305 publications containing 576 case reports, results showed that human dirofilariosis is currently caused by 5 Dirofilaria species (mainly D. repens). Maturation was not uncommon, since 42.95% of the parasites recovered were described as mature worms, most of them females, 26.42% of which contained micofilariae in the uterus. Moreover, 6 microfilaremic cases have been described. The predominant clinical manifestation was the presence of a worm encapsulated within a nodule, but there is a considerable variety of accompanying symptoms depending on anatomical location and type of dirofilariosis. Parasites/nodules were found in 71 different anatomical locations, being the traditional nomenclature of human dirofilariosis unable to properly cover this complex situation. Delay in seeking medical assistance (patient perception) and the frequency of wrong clinical suspicions (doctor knowledge), strongly influenced clinical management. The initial suspicion in cases of subcutaneous and pulmonary dirofilariosis is predominantly a tumour, while in the ocular dirofilariosis a parasite (but not directly Dirofilaria) is mostly suspected. Surgery is usually applied, regardless of the use of non‐invasive techniques during preoperative management and although its use is still limited, molecular approach is the most accurate technique to establish a species‐level diagnosis. Accurate epidemiological, parasitological and clinical information while handling and reporting human clinical cases is a need for physicians and researchers to improve and standardize the clinical management of human dirofilariosis. This article is protected by copyright. All rights reserved

Simple Summary Ascaris suum parasitises pigs all over the world causing a disease responsible for producing reductions in weight gains and damages to several organs of the infected animals that incur huge economic losses for the swine industry. While adult worms of this parasite are located in the small intestine of the host, their larval stages migrate through the bloodstream as an evolutionary advantageous strategy within a hostile environment that confronts host responses such as blood clots formation. The aim of this work is to study the ability of A. suum larvae to inhibit blood coagulation as a possible mechanism to control blood clots formation and facilitate their migration. The results showed that these larvae inhibited host blood coagulation and possessed molecules similar to those responsible for inhibiting blood coagulation in pigs. The anticoagulant effect of A. suum larvae could constitute a potential survival mechanism for the parasite. Therefore, developing new control strategies directed at this and similar processes could avoid A. suum larval migration and the establishment of adult worms in their definitive location, which is necessary to confront the damages and economic losses produced by this parasitosis. Abstract In order to evade the response of their hosts, helminth parasites have evolved precise and highly regulated mechanisms, including migration strategies of the larval stages. In regard to porcine ascariosis caused by Ascaris suum, its infective third-stage larvae (AsL3) undergo a complex migratory route through the bloodstream of their host before establishing in the small intestine to reach maturation. Despite the benefits attributed to this migration, blood clots formation could compromise larvae survival. The aim of this work was to study the interaction between the cuticle and excretory/secretory antigens of AsL3 and the host coagulation cascade. Larvae were obtained after incubating and hatching A. suum eggs, after which the antigenic extracts were produced. Their ability to disrupt the coagulation cascade was studied using anticoagulation and chromogenic assays, and techniques based on electrophoresis. The obtained results showed that both antigenic extracts possessed anticoagulant potential, being able to inhibit the intrinsic, extrinsic and/or common pathways of the blood coagulation cascade as well as the activated factor X. Moreover, three A. suum serpin proteins were identified as candidates to inhibit this host coagulation factor. To the best of our knowledge, this study shows, for the first time, the anticoagulant potential of the infective larvae of A. suum, which could be used by the parasite as a mechanism to facilitate its invasion and survival in the host.

The objective of this work was to determine the risk of exposure to Dirofilaria immitis in the stray dog population and people living in the metropolitan area of Puebla City, Mexico, using serologic methods. A total of 283 blood salmples were collected from dogs and 254 salmples from people. The canine samples were analyzed using a commercial kit to detect D. immitis antigens and the Knott test to detect microfilariae. Human samples were analyzed by ELISAs to detect antibodies against D. immitis and Wolbachia anti-WSP, as well as Western blot to identify specific bands of the parasite in the adult antigenic extract of D. immitis. Positive results were represented on a GIS map that included layers with geoenvironmental information relevant to parasite transmission in the study area. The prevalence of D. immitis in the canine stray population was 2.12 %, with positive cases found in individuals aged 1–10 years. Human seroprevalence was established at 5.11 %, with women and men similarly affected. By age, a significantly higher seroprevalence was observed in younger individuals (<18 years) than in the rest of the population. The geolocation of the results showed that positive cases were found in the environment (within 2 km) of potentially favorable areas for mosquito breeding. The importance of human influence in the creation and maintenance of favorable conditions for the transmission of canine and human dirofilariosis within an urban area is highlighted, both by the lack of environmental sanitation and by the absence of measures to limit or eliminate the canine reservoir population.