Identification and Characterization of Three New Antimicrobial Peptides from the Marine Mollusk Nerita versicolor (Gmelin, 1791).

Mollusks have been widely investigated for antimicrobial peptides because their humoral defense against pathogens is mainly based on these small biomolecules. In this report, we describe the identification of three novel antimicrobial peptides from the marine mollusk Nerita versicolor. A pool of N. versicolor peptides was analyzed with nanoLC-ESI-MS-MS technology, and three potential antimicrobial peptides (Nv-p1, Nv-p2 and Nv-p3) were identified with bioinformatical predictions and selected for chemical synthesis and evaluation of their biological activity. Database searches showed that two of them show partial identity to histone H4 peptide fragments from other invertebrate species. Structural predictions revealed that they all adopt a random coil structure even when placed near a lipid bilayer patch. Nv-p1, Nv-p2 and Nv-p3 exhibited activity against Pseudomonas aeruginosa. The most active peptide was Nv-p3 with an inhibitory activity starting at 1.5 µg/mL in the radial diffusion assays. The peptides were ineffective against Klebsiella pneumoniae, Listeria monocytogenes and Mycobacterium tuberculosis. On the other hand, these peptides demonstrated effective antibiofilm action against Candida albicans, Candida parapsilosis and Candida auris but not against the planktonic cells. None of the peptides had significant toxicity on primary human macrophages and fetal lung fibroblasts at effective antimicrobial concentrations. Our results indicate that N. versicolor-derived peptides represent new AMP sequences and have the potential to be optimized and developed into antibiotic alternatives against bacterial and fungal infections.

Immunological Resistance of Pseudosuccinea columella Snails From Cuba to Fasciola hepatica (Trematoda) Infection: What We Know and Where We Go on Comparative Molecular and Mechanistic Immunobiology, Ecology and Evolution.

One of the most interesting biological models is that of snail-trematode interactions, many of which ultimately result in the transmission of several important diseases, particularly in the tropics. Herein, we review the scientific advances on a trematode-snail system in which certain populations of Pseudosuccinea columella (a common host species for trematodes) have been demonstrated naturally-resistant to Fasciola hepatica, in association with an effective encapsulation of the parasite by innate immune cells of the host, the hemocytes. Emphasis is made on the molecular and immunological features characterizing each P. columella phenotype in relation to their anti-parasitic competence, their distinctive ecological patterns and the existence of a significant cost of resistance. An integrative overview of the resistance to F. hepatica through comparative immunobiology, genetics and ecology is presented to hypothesize on the possible origins and evolution of this phenomenon and to postulate significant roles for parasite mediated-selection and environmental factors in shaping and maintaining the resistant phenotype in the field. Lastly, clues into future experimental perspectives to deeply characterize the interplay between P. columella and F. hepatica and the immunobiology of the resistance are also included. The advances revised in the present paper are only beginning to unravel mechanisms of anti-parasite innate defense responses and their evolutionary bases, and can facilitate the development of prospective approaches towards practical applications of P. columella resistance.

On the arrival of fasciolosis in the Americas.

Fasciola hepatica is a worldwide emerging and re-emerging parasite heavily affecting several regions in South America. Some lymnaeid snail species of American origin are among the major hosts of F. hepatica worldwide. Recent paleoparasitological findings detected its DNA in a 2300-year-old sample in Patagonia, countering the common hypothesis of the recent arrival of F. hepatica in the Americas during European colonization. Thus, the theory of an initial introduction in the 1500s can no longer be sustained. This article discusses how it was possible for F. hepatica to reach and spread in the Americas in relation to the availability and compatibility of hosts through natural and incidental introductions. Our study will serve to better understand the ongoing Neotropical scenario of fasciolosis.

Antimicrobial Peptides Pom-1 and Pom-2 from Pomacea poeyana Are Active against Candidaauris, C. parapsilosis and C. albicans Biofilms.

Recently two peptides isolated from the Cuban freshwater snail Pomacea poeyana (Pilsbry, 1927) were described to have antimicrobial activity against bacterial pathogens. Here we show considerable activities of Pom-1 and Pom-2 to reduce the viability of C. albicans, C. parapsilosis and the less common species C. auris measured as the decrease of metabolic activity in the resazurin reduction assay for planktonic cells. Although these activities were low, Pom-1 and Pom-2 turned out to be highly potent inhibitors of biofilm formation for the three Candida species tested. Whereas Pom-1 was slightly more active against C. albicans and C. parapsilosis as representatives of the more common Candida species Pom-2 showed no preference and was fully active also against biofilms of the more uncommon species C. auris. Pom-1 and Pom-2 may represent promising lead structures for the development of a classical peptide optimization strategy with the realistic aim to further increase antibiofilm properties and other pharmacologic parameters and to generate finally the first antifungal drug with a pronounced dedication against Candida biofilms.

Genetic diversity and relationships of the liver fluke Fasciola hepatica (Trematoda) with native and introduced definitive and intermediate hosts.

Fasciolosis is a worldwide spread parasitosis mainly caused by the trematode Fasciola hepatica. This disease is particularly important for public health in tropical regions, but it can also affect the economies of many developed countries due to large infections in domestic animals. Although several studies have tried to understand the transmission by studying the prevalence of different host species, only a few have used population genetic approaches to understand the links between domestic and wildlife infections. Here, we present the results of such genetic approach combined with classical parasitological data (prevalence and intensity) by studying domestic and wild definitive hosts from Camargue (southern France) where fasciolosis is considered as a problem. We found 60% of domestic hosts (cattle) infected with F. hepatica but lower values in wild hosts (nutria, 19%; wild boars, 4.5%). We explored nine variable microsatellite loci for 1,148 adult flukes recovered from four different populations (non-treated cattle, treated cattle, nutria and wild boars). Populations from the four groups differed, though we found a number of migrants particularly non-treated cattle and nutria. Overall, we detected 729 different multilocus genotypes (from 783 completely genotyped individuals) and only 46 genotypes repeated across samples. Finally, we experimentally infected native and introduced intermediate snail hosts to explore their compatibility with F. hepatica and assess the risks of fasciolosis expansion in the region. The introduced species Galba truncatula and Pseudosuccinea columella attained the higher values of overall compatibility in relation to the European species. However, concerning the origin, sympatric combinations of G. truncatula were more compatible (higher prevalence, intensity and survival) than the allopatric tested. According to our results, we should note that the assessment of epidemiological risks cannot be limited to a single host-parasite system, but should focus on understanding the diversity of hosts in the heterogeneous environment through space and time.

Towards the comprehension of fasciolosis (re-)emergence: an integrative overview.

The increasing distribution and prevalence of fasciolosis in both human and livestock are concerning. Here, we examine the various types of factors influencing fasciolosis transmission and burden and the interrelations that may exist between them. We present the arsenal of molecules, 'adjusting' capabilities and parasitic strategies of Fasciola to infect. Such features define the high adaptability of Fasciola species for parasitism that facilitate their transmission. We discuss current environmental perturbations (increase of livestock and land use, climate change, introduction of alien species and biodiversity loss) in relation to fasciolosis dynamics. As Fasciola infection is directly and ultimately linked to livestock management, living conditions and cultural habits, which are also changing under the pressure of globalization and climate change, the social component of transmission is also discussed. Lastly, we examine the implication of increasing scientific and political awareness in highlighting the current circulation of fasciolosis and boosting epidemiological surveys and novel diagnostic techniques. From a joint perspective, it becomes clear that factors weight differently at each place and moment, depending on the biological, environmental, social and political interrelating contexts. Therefore, the analyses of a disease as complex as fasciolosis should be as integrative as possible to dissect the realities featuring each epidemiological scenario. Such a comprehensive appraisal is presented in this review and constitutes its main asset to serve as a fresh integrative understanding of fasciolosis.

New Antibacterial Peptides from the Freshwater Mollusk Pomacea poeyana (Pilsbry, 1927).

Antimicrobial peptides (AMPs) are biomolecules with antimicrobial activity against a broad group of pathogens. In the past few decades, AMPs have represented an important alternative for the treatment of infectious diseases. Their isolation from natural sources has been widely investigated. In this sense, mollusks are promising organisms for the identification of AMPs given that their immune system mainly relies on innate response. In this report, we characterized the peptide fraction of the Cuban freshwater snail Pomacea poeyana (Pilsbry, 1927) and identified 37 different peptides by nanoLC-ESI-MS-MS technology. From these peptide sequences, using bioinformatic prediction tools, we discovered two potential antimicrobial peptides named Pom-1 (KCAGSIAWAIGSGLFGGAKLIKIKKYIAELGGLQ) and Pom-2 (KEIERAGQRIRDAIISAAPAVETLAQAQKIIKGG). Database search revealed that Pom-1 is a fragment of Closticin 574 previously isolated from the bacteria Clostridium tyrobutyrium, and Pom-2 is a fragment of cecropin D-like peptide first isolated from Galleria mellonella hemolymph. These sequences were chemically synthesized and evaluated against different human pathogens. Interestingly, structural predictions of both peptides in the presence of micelles showed models that comprise two alpha helices joined by a short loop. The CD spectra analysis of Pom-1 and Pom-2 in water showed for both structures a high random coil content, a certain content of α-helix and a low ß-sheet content. Like other described AMPs displaying a disordered structure in water, the peptides may adopt a helical conformation in presence of bacterial membranes. In antimicrobial assays, Pom-1 demonstrated high activity against the Gram-negative bacteria Pseudomonas aeruginosa and moderate activity against Klebsiella pneumoniae and Listeria monocytogenes. Neither of the two peptides showed antifungal action. Pom-1 moderately inhibits Zika Virus infection but slightly enhances HIV-1 infectivion in vitro. The evaluation of cell toxicity on primary human macrophages did not show toxicity on THP-1 cells, although slight overall toxicity was observed in high concentrations of Pom-1. We assume that both peptides may play a key role in innate defense of P. poeyana and represent promising antimicrobial candidates for humans.

The immunobiological interplay between Pseudosuccinea columella resistant/susceptible snails with Fasciola hepatica: Hemocytes in the spotlight.

The Fasciola hepatica/Pseudosuccinea columella interaction in Cuba involves a unique pattern of phenotypes; while most snails are susceptible, some field populations are naturally resistant to infection and parasites are encapsulated by snail hemocytes. Thus, we investigated the hemocytes of resistant (R) and susceptible (S) P. columella, in particular morphology, abundance, proliferation and in vitro encapsulation activity following exposure to F. hepatica. Compared to susceptible P. columella, hemocytes from exposed resistant snails showed increased levels of spreading and aggregation (large adherent cells), proliferation of circulating blast-like cells and encapsulation activity of the hemocytes, along with a higher expression of the cytokine granulin. By contrast, there was evidence of a putative F. hepatica-driven inhibition of host immunity, only in susceptible snails. Additionally, (pre-)incubation of naïve hemocytes from P. columella (R and S) with different monosaccharides was associated with lower encapsulation activity of F. hepatica larvae. This suggests the involvement in this host-parasite interaction of lectins and lectins receptors (particularly related to mannose and fucose sensing) in association with hemocyte activation and/or binding to F. hepatica.

Improving the sensitivity of an hsp20-based PCR for genus detection of Leishmania parasites in cutaneous clinical samples: a proof of concept.

Leishmaniasis is a parasitic disease of medical importance widely distributed around the world. Several methods are available for diagnosis but molecular approaches are highly recommended. To improve the sensitivity of an existing hsp20 gene based-PCR protocol to detect Leishmania parasites, primers were redesigned to amplify a shorter fragment using a new PCR variant (PCR-hsp20S). In this study, we aimed at characterizing the performance of the new method on cutaneous clinical samples and compare it with the former PCR-hsp20. The analytical sensitivity of the PCR-hsp20S was evaluated using DNA dilutions (100-0.1 pg) from Leishmania donovani and resulted in the detection of 10 fg of parasitic DNA, the equivalent to 0.05 parasite genome. For the diagnostic evaluation, a panel of 127 human clinical samples was used to calculate the parameters of sensitivity, specificity, accuracy, and positive and negative predictive values of the PCR-hsp20S. Diagnostic sensitivity was 94% (CI, 89.1-99.7%) and the specificity of 100% (CI, 98.6-100%). The same panel was also evaluated with the PCR-hsp20 to calculate the agreement between both molecular assays and to compare their performances. While both hsp20-based PCRs showed a good agreement coefficient (kappa index = 0.6), the performance of the novel variant, PCR-hsp20S, was significantly higher in terms of sensitivity (P = 0.0001) allowing the accurate detection of a higher number of Leishmania-positive clinical samples. We endorse the use of the PCR-hsp20S over the former protocol for the detection of Leishmania parasites from cutaneous clinical samples. In addition, as an improved sensitivity was achieved with the new method merely through the amplification of a shorter gene fragment, this investigation constitutes an experimental proof of this concept.

Patterns of distribution, population genetics and ecological requirements of field-occurring resistant and susceptible Pseudosuccinea columella snails to Fasciola hepatica in Cuba.

Pseudosuccinea columella snails transmit the trematode Fasciola hepatica, but in Cuba, six naturally occurring populations successfully resist parasite infection. Here, we present an updated distribution of P. columella in Cuba; 68 positive sites with the earliest records more abundant in west-central Cuba and with east-central populations generally corresponding to the newest samples. No records were found farther east. The IPA site reported 10.5% prevalence of F. hepatica-infected snails. Population genetics, studied through microsatellites, showed low allelic and multilocus genotypic richness (MLGT), mainly in susceptible populations, strong deviations from panmixia and high self-fertilization rates. Susceptible individuals were grouped in one major cluster containing the majority of MLGT, and two independent clusters grouped the MLGT of resistant individuals from western and central populations, respectively. From these, we propose that several introductions of P. columella occurred in Cuba, primarily in the west, with the early arrivals deriving on the resistant populations. A more recent introduction of susceptible P. columella carrying MLGT T and Y may have occurred, where the latter spread quickly through the island and possibly increase the risk of parasite transmission in Cuba since all snails naturally infected with F. hepatica were carriers of the MLGT Y. Interestingly, even though resistant populations are highly diverse and are likely the oldest within Cuba, they are only found in six localities characterized by soft (total hardness, TH = 6.3 ± 1.03°d) and slightly acidic (pH = 6.2 ± 0.12) waters with low richness in snail species (3.2 ± 1.02). This tendency was also observed in a two-year follow-up ecological study that was conducted on a farm where both phenotypes occurred in sympatry; colonization events by resistant over susceptible snails coincided with a reduction in the pH and TH of the water. A comparison of life traits in susceptible and resistant isolates reared at two different pH/TH conditions (5.9/4°d or 7.8/14°d) showed that low pH/TH negatively affects P. columella, irrespective of the phenotype. However, evidence of higher tolerance (higher survival, life expectancy, egg viability) to such conditions was observed in resistant isolates. Finally, we speculate that the limited distribution of resistant populations might be related to a better exploitation of sites that are less suitable to snails (thus, with lower competition), rather than to a differential ecological restriction to specific environmental conditions from susceptible P. columella.

Reviewing Fasciola hepatica transmission in the West Indies and novel perceptions from experimental infections of sympatric vs. allopatric snail/fluke combinations.

Fasciolosis is an important food-borne parasitic disease affecting over two million people worldwide with economic losses related to cattle production of up to US$ 3 billion annually. Despite the long known presence of Fasciola hepatica in the Caribbean islands its transmission is not well known. This study reviews historical and recent data on fasciolosis in the West Indies, revealing for the first time the outcomes of sympatric and allopatric fluke/snail interactions in the area by exploring the susceptibility of four lymnaeid species after exposure to F. hepatica isolates from Cuba, the Dominican Republic and France. Overall, Galba cubensis showed a mean prevalence of 71.8% and appears to be the most suitable intermediate host species irrespective of the isolate used. Sympatric combinations (snail and parasite from the same country) were generally more compatible (higher susceptibility, parasite intensity and snail survival post-exposure) and only the allopatric interaction of French G. truncatula/Cuban F. hepatica attained 100% prevalence and mean intensity over 33 rediae/snail. However, certain Dominican populations of Pseudosuccinea columella showed high parasite intensities (>30 rediae/snail) when infected with Cuban flukes, highlighting the potential risks of biological introductions. Overall, high compatibility in most sympatric combinations compared to low or moderate compatibility in allopatric ones, suggests the existence of local adaptation from a long sustained interaction that has led to high rates of transmission. Interestingly, attempts to infect G. schirazensis with sympatric and allopatric flukes failed and coupled with the lowest survival rates which supposes a low risk of fasciolosis transmission in areas where this is the only snail species. Although there are significant gaps in the actual status of fasciolosis transmission from several islands in the West Indies these results show a permanent risk. We conclude that fasciolosis transmission is high in areas where the local snail, G. cubensis, occurs, and will be even higher in the presence of the invasive P. columella.

Natural resistance to Fasciola hepatica (Trematoda) in Pseudosuccinea columella snails: A review from literature and insights from comparative "omic" analyses.

The snail Pseudosuccinea columella is one of the main vectors of the medically-important trematode Fasciola hepatica. In Cuba, the existence of natural P. columella populations that are either susceptible or resistant to F. hepatica infection offers a unique snail-parasite for study of parasite-host compatibility and immune function in gastropods. Here, we review all previous literature on this system and present new "omic" data that provide a molecular baseline of both P. columella phenotypes from naïve snails. Comparison of whole snail transcriptomes (RNAseq) and the proteomes of the albumen gland (2D-electrophoresis, MS) revealed that resistant and susceptible strains differed mainly in an enrichment of particular biological processes/functions and a greater abundance of proteins/transcripts associated with immune defense/stress response in resistant snails. These results indicate a differential allocation of molecular resources to self-maintenance and survival in resistant P. columella that may cause enhanced responsiveness to stressors (i.e. F. hepatica infection or tolerance to variations in environmental pH/total water hardness), possibly as trade-off against reproduction and the ecological cost of resistance previously suggested in resistant populations of P. columella.

Genetic Diversity of Trichomonas Vaginalis Clinical Isolates According to Restriction Fragment Length Polymorphism Analysis of the 60-kDa Proteinase Gene.

INTRODUCCTION: Trichomonas vaginalis is a highly prevalent parasitic that causes the sexually transmitted disease trichomoniasis with some serious health complications. More understanding about genetic features of the parasite can be helpful in the study of the pathogenesis, epidemiology of the infection and drug susceptibility. For this end, we conducted analysis of a fragment (23 kDa) of the p60 of T. vaginalis gene. MATERIAL AND METHODS: The restriction fragment length polymorphism (RFLP) methods was used. RESULT AND DISCUSSION: RFLP analysis showed the difference between T. vaginalis isolates from symptomatic and asymptomatic patients, suggesting a relation between the genetic identity of the isolates and their clinical manifestations.

Fasciola hepatica-Pseudosuccinea columella interaction: effect of increasing parasite doses, successive exposures and geographical origin on the infection outcome of susceptible and naturally-resistant snails from Cuba.

BACKGROUND: Pseudosuccinea columella is one of the most widespread vectors of Fasciola hepatica, a globally distributed trematode that affects humans, livestock and wildlife. The exclusive occurrence in Cuba of susceptible and naturally-resistant populations to F. hepatica within this snail species, offers a fascinating model for evolutionary biology, health sciences and vector control strategies. In particular, resistance in P. columella is characterized by the encapsulation of the parasite by host's immune cells and has been experimentally tested using different Cuban F. hepatica isolates with no records of successful infection. Here, we aimed to explore for the first time, the effect of different parasite doses, successive exposures and different parasite origins on the infection outcomes of the two phenotypes of P. columella occurring in Cuba. METHODS: To increase the chances for F. hepatica to establish, we challenged Cuban P. columella with increasing single parasite doses of 5, 15 or 30 miracidia and serial exposures (three-times) of 5 miracidia using a sympatric F. hepatica isolate from Cuba, previously characterized by microsatellite markers. Additionally, we exposed the snails to F. hepatica from different geographical origins (i.e. Dominican Republic and France). Parasite prevalence, redial burden and survival of snails were recorded at 25 days post-exposure. RESULTS: No parasite development was noted in snails from the resistant populations independent of the experimental approach. Contrastingly, an overall increase in prevalence and redial burden was observed in susceptible snails when infected with high miracidia doses and after serial exposures. Significant differences in redial burden between single 15 miracidia and serial 3 × 5 miracidia infected snails suggest that immune priming potentially occurs in susceptible P. columella. Compatibility differences of allopatric (Caribbean vs European) F. hepatica with susceptible snails were related to the geographical scale of the combinations. CONCLUSIONS: Here, the effectiveness of P. columella resistance to F. hepatica does not decline with increasing parasite doses, successive infection or different geographical origins of parasite isolates, while presenting new evidence for specificity for infection in susceptible P. columella snails. Understanding the peculiarities of the P. columella-F. hepatica interaction and the extent of the resistant phenotype is crucial for an effective parasite control and for developing alternatives to tackle fasciolosis transmission.

Updated distribution and experimental life-history traits of the recently invasive snail Lissachatina fulica in Havana, Cuba.

The giant African snail Lissachatina fulica has been reported invading Cuba since 2014 and is now well established in areas of Havana and several nearby regions. This invasive species is of major concern to health authorities given its role as an important vector of parasites such as Angiostrongylus cantonensis, the causative agent of eosinophilic meningoencephalitis in humans. Here, we update the distribution within Cuba. We also report on our studies of experimental life-history traits to assess the population dynamics and potential for spread of this species in Cuba. The experimental population had a very low probability of dying at first age intervals (Type I survival curve) with a life expectancy of 71 weeks. During our experiment, sexual maturity was reached after 22 weeks (individuals of 70 mm shell length) and eggs were laid regularly (mean batch size: 188 ±â€¯111.79 SD). We failed to detect any deviations from the mortality curve and individuals reached an average of 77.3 mm (shell length) and weighted 57.7 g after one year. Predicted curve models indicate that snails reaching their average lifespan of five years should attain 10-12 cm (shell length) and weight 160 g. The spreading of this invasive and vector snail has been tracked for four years in Cuba showing a steady increase of invaded localities. How fast and how far this species develops in Cuba is unknown but the life history parameters indicated in this paper show that it has a large potential to invade all areas of Cuba quickly unless a systematic abatement strategy is developed.

Detected trematodes inside blue-winged teals (Spatula discors) give insights on north-south flow of parasites through Cuba during migration.

The Blue-winged Teal (Spatula discors) is known for engaging in long-distance North-South migrations and back in the Americas with Cuba being an important wintering ground. Teals serve as hosts for a wide range of parasite species that can be "discharged" at each end of the migration route. Here, we explore for the first time the diversity of trematodes that the Blue-winged Teal may be introducing to -or exporting from- Cuba. We found 15 digenean parasites in 65 sampled teals of which 13 represent first reports for Cuba with one species (Echinostoma revolutum) of zoonotic importance. Overall prevalence was very high (96.92%) whereas Trichobilharzia spp. resulted the most ubiquitous parasite. Highest intensities were recorded for Australapatemon sp. (arriving teals) and Levinseniella amnicolae (departing teals). Altogether, departing teals harboured the highest number of trematode species and abundance. The short development and lifespan, inside migratory birds, of adult stages of trematodes like Trichobilharzia sp., Cotylurus flabelliformis and Microphallus pygmaeus strongly suggest that the occurrence of these trematodes in the departing teals could be related to their long establishment in Cuban ecosystems. In a global scenario where parasitology, particularly wildlife parasitology, is mostly overlooked, there should be an increasing need of gathering information and increasing surveillance of wildlife diseases that might eventually become important for the health of ecosystems and of domestic animals and humans. The present study constitutes the first major attempt to explore trematode infection from S. discors in Cuba under the view of parasite flow via bird migration.

Detection and identification of Leishmania spp.: application of two hsp70-based PCR-RFLP protocols to clinical samples from the New World.

Leishmaniasis is highly prevalent in New World countries, where several methods are available for detection and identification of Leishmania spp. Two hsp70-based PCR protocols (PCR-N and PCR-F) and their corresponding restriction fragment length polymorphisms (RFLP) were applied for detection and identification of Leishmania spp. in clinical samples recruited in Colombia, Guatemala, and Honduras. A total of 93 cases were studied. The samples were classified into positive or suspected of leishmaniasis according to parasitological criteria. Molecular amplification of two different hsp70 gene fragments and further RFLP analysis for identification of Leishmania species was done. The detection in parasitologically positive samples was higher using PCR-N than PCR-F. In the total of samples studied, the main species identified were Leishmania panamensis, Leishmania braziliensis, and Leishmania infantum (chagasi). Although RFLP-N was more efficient for the identification, RFLP-F is necessary for discrimination between L. panamensis and Leishmania guyanesis, of great importance in Colombia. Unexpectedly, one sample from this country revealed an RFLP pattern corresponding to Leishmania naiffi. Both molecular variants are applicable for the study of clinical samples originated in Colombia, Honduras, and Guatemala. Choosing the better tool for each setting depends on the species circulating. More studies are needed to confirm the presence of L. naiffi in Colombian territory.

Insights into the biological features of the antigenic determinants recognized by four monoclonal antibodies in redia and adult stages of the liver fluke Fasciola hepatica.

Fasciola hepatica is a digenean trematode which infects a wide variety of domestic animals and also humans. Previous studies have demonstrated that four monoclonal antibodies (Mabs) against the total extract of F. hepatica redia (named as 1E4, 6G11, 4E5 and 4G11) also recognized the excretion - secretion antigens (ES Ag) of adult parasites, which is a biologically-relevant mixture of molecules with functional roles during infection and immune evasion on definitive hosts. In the present report we describe the partial characterization of the epitopes recognized by these Mabs by heat treatment, mercaptoethanol reduction, pronase proteolysis and sodium peryodate oxidation, which suggested their predominant protein and conformational nature. Also, a comparative study using immunodetection assays on crude extracts and on histological sections of both rediae and adults of F. hepatica were performed to explore the expression pattern of the antigenic determinants in these developmental stages. From these experiments it was found that the Mabs reacted most likely with the same proteins of approximately 64 and 105 kDa present on both rediae and adult's extracts. However, the 1E4, 6G11 and 4E5 Mabs also recognized other molecules of the total extract of F. hepatica adults, a fact that constitutes an evidence of the antigenic variation between both stages and points at a certain biological relevance of the recognized antigenic determinants. Immunolocalization studies on histological sections revealed that all Mabs reacted with the tegument of F. hepatica in both rediae and adults stages, while the epitopes recognized by 1E4, 6G11 and 4E5 antibodies were also preferentially localized in the intestinal caeca and in different organs of the reproductive system of adult specimens. The immunogenicity of these antigenic determinants, their conserved status among different stages of the life cycle of F. hepatica and their presence in both tegument and ES Ag of adult parasites, are suitable features that suggest their potential use for developing an epitope-based vaccine for fasciolosis control.

Isolation of 1E4 IgM Anti-Fasciola hepatica Rediae Monoclonal Antibody from Ascites: Comparison of Two Purification Protocols.

Purification of immunoglobulin M (IgM) antibodies could be challenging, and is often characterized by the optimization of the purification protocol to best suit the particular features of the molecule. Here, two different schemes were compared to purify, from ascites, the 1E4 IgM monoclonal antibody (mAb) previously raised against the stage of redia of the trematode Fasciola hepatica. This immunoglobulin is used as capture antibody in an immunoenzymatic assay to detect parasite ongoing infection in its intermediate hosts. The first purification protocol of the 1E4 mAb involved two chromatographic steps: an affinity chromatography on a Concanavalin A matrix followed by size exclusion chromatography. An immunoaffinity chromatography was selected as the second protocol for one-step purification of the antibody using the crude extract of adult parasites coupled to a commercial matrix. Immunoreactivity of the fractions during purification schemes was assessed by indirect immunoenzymatic assays against the crude extract of F. hepatica rediae, while purity was estimated by protein electrophoresis. Losses on the recovery of the antibody isolated by the first purification protocol occurred due to protein precipitation during the concentration of the sample and to low resolution of the size exclusion molecular chromatography step regarding this particular immunoglobulin. The immunoaffinity chromatography using F. hepatica antigens as ligands proved to be the most suitable protocol yielding a pure and immunoreactive antibody. The purification protocols used are discussed regarding efficiency and difficulties.